Streptococcus suis vaccines and diagnostic tests

ABSTRACT

Described are  Streptococcus suis  infections in pigs, vaccines directed against those infections and tests for diagnosing  S. suis  infections. Provided is an isolated or recombinant nucleic acid encoding a capsular gene cluster of  S. suis  or a gene or gene fragment derived therefrom. Further provided is a nucleic acid probe or primer allowing species or serotype-specific detection of  S. suis . Also provided is a  Streptococcus suis  antigen and vaccine derived therefrom.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of co-pending U.S. patent applicationSer. No. 11/516,691, filed Sep. 5, 2006, which is a continuation of U.S.patent application Ser. No. 09/767,041, filed on Jan. 22, 2001, now U.S.Pat. No. 7,125,548, issued Oct. 24, 2006, which is a continuation ofInternational Application No. PCT/NL99/00460, filed on Jul. 19, 1999,designating the United States of America, the PCT International PatentApplication itself claiming priority from European Patent OfficeApplication Serial No. 98202465.5 filed Jul. 22, 1998 and EuropeanPatent Office Application Serial No. 98202467.1 filed Jul. 22, 1998, thecontents of each of which are incorporated herein by this reference.

STATEMENT ACCORDING TO 37 C.F.R. §1.52(e)(5) Sequence Listing Submittedon Compact Disc

Pursuant to 37 C.F.R. §1.52(e)(1)(iii), a compact disc containing anelectronic version of the Sequence Listing has been submittedconcomitant with this application, the contents of which are herebyincorporated by reference. A second compact disc is submitted and is anidentical copy of the first compact disc. The discs are labeled “copy 1”and “copy 2,” respectively, and each disc contains one file entitled“Sequence Listing.txt” which is 130 KB and created on Sep. 5, 2006.

TECHNICAL FIELD

The invention relates to Streptococcus infections in pigs, vaccinesdirected against those infections, tests for diagnosing Streptococcusinfections and bacterial vaccines. More particularly, the inventionrelates to vaccines directed against Streptococcus infections.

BACKGROUND

Streptococcus species, of which a large variety causes infections indomestic animals and man, are often grouped according to Lancefield'sgroups. Typing according to Lancefield occurs on the basis ofserological determinants or antigens that are, among others, present inthe capsule of the bacterium, and allows for only an approximatedetermination. Often, bacteria from different groups showcross-reactivity with each other, while other Streptococci cannot beassigned a group-determinant at all. Within groups, furtherdifferentiation is often possible on the basis of serotyping. Theseserotypes further contribute to the large antigenic variability ofStreptococci, a fact that creates an array of difficulties withindiagnosis of and vaccination against Streptococcal infections.

Lancefield group A Streptococcus species (Group A Streptococci “GAS,”Streptococcus pyogenes) are common in children, causing nasopharyngealinfections and complications thereof. Among animals, cattle areespecially susceptible to GAS infection which can cause mastitis.

Group A streptococci are the etiologic agents of streptococcalpharyngitis and impetigo, two of the most common bacterial infections inchildren, as well as a variety of less common, but potentiallylife-threatening, infections including soft tissue infections,bacteremia, and pneumonia. In addition, GAS are uniquely associated withthe post-infectious autoimmune syndromes of acute rheumatic fever andpost streptococcal glomerulonephritis.

Several recent reports suggest that the incidence of both seriousinfections due to GAS and acute rheumatic fever has increased during thepast decade, focusing renewed interest on defining the attributes orvirulence factors of the organism that may play a role in thepathogenesis of these diseases.

GAS produce several surface components and extracellular products thatmay be important in virulence. The major surface protein, M protein, hasbeen studied in the most detail and has been convincingly shown to playa role in both virulence and immunity. Isolates rich in M protein areable to grow in human blood, a property thought to reflect the capacityof N protein to interfere with phagocytosis, and these isolates tend tobe virulent in experimental animals.

Lancefield group B Streptococcus (“GBS”) are most often seen in cattle,causing mastitis; however, human infants are susceptible as well, oftenwith fatal consequences. Group B streptococci (GBS) constitute a majorcause of bacterial sepsis and meningitis among human neonates born inthe United States and Western Europe and are emerging as significantneonatal pathogens in developing countries as well.

It is estimated that GBS strains are responsible for 10,000 to 15,000cases of invasive infection in neonates in the United States alone.Despite advances in early diagnosis and treatment, neonatal sepsis dueto GBS continues to carry a mortality rate of 15 to 20%. In addition,survivors of GBS meningitis have 30 to 50% incidence of long-termneurologic sequelae. Over the past two decades, increasing recognitionof GBS as an important pathogen for human infants has generated renewedinterest in defining the bacterial and host factors important invirulence of GBS and in the immune response to GBS infection.

Particular attention has focused on the capsular polysaccharide as thepredominant surface antigen of the organisms. In a modification of thesystem originally developed by Rebecca Lancefield, GBS strains areserotyped on the basis of antigenic differences in their capsularpolysaccharides and the presence or absence of serologically defined Cproteins. While GBS isolated from non-human sources often lack aserologically detectable capsular, a large majority of strainsassociated with neonatal infection belong to one of four major capsularserotypes, 1a, 1 b, II or III. The capsular polysaccharide forms theoutermost layer around the exterior of the bacterial cell, superficialto the cell wall. The capsule is distinct from the cell wall-associatedgroup B carbohydrate. It has been suggested that the presence of sialicacid, in the capsule of bacteria that causes meningitis, is importantfor allowing these bacteria to breach the blood-brain barrier. Indeed,in S. agalactiae, sialic acid has been shown to be critical for thevirulence function of the type III capsule. The capsule of S. suisserotype is composed of glucose, galactose, N-acetylglucosamine,rhamnose and sialic acid.

The group B polysaccharide, in contrast to the type-specific capsule, ispresent on all GBS strains and is the basis for serogrouping theorganisms into Lancefield's group B. Early studies by Lancefield andco-workers showed that antibodies raised in rabbits against whole GBSorganisms protected mice against challenge with strains of homologouscapsular type, demonstrating the central role of the capsularpolysaccharide as a protective antigen. Studies in the 1970s by Bakerand Kasper demonstrated that cord blood of human infants with type IIIGBS sepsis uniformly had low or undetectable levels of antibodiesdirected against the type III capsule, suggesting that a deficiency ofanticapsular antibody was a key factor in susceptibility of humanneonates to GBS disease.

Lancefield group C infections, such as those with S. equi, S.zooepidemicus, S. dysgalactiae, and others, are mainly seen in horses,cattle and pigs, but can also cross the species barrier to humans.Lancefield group D (S. bovis) infections are found in all mammals andsome birds, sometimes resulting in endocarditis or septicemia.

Lancefield groups E, G, L, P, U and V (S. porcinus, s. canis, s.dysgalactiae) are found in various hosts, causing neonatal infections,nasopharyngeal infections or mastitis.

Within Lancefield groups R, S and T (and with ungrouped types),Streptococcus suis is an important cause of meningitis, septicemia,arthritis and sudden death in young pigs (4, 46). Incidentally, it canalso cause meningitis in man (1). S. suis strains are usually identifiedand classified by their morphological, biochemical and serologicalcharacteristics (58, 59, 46). Serological classification is based on thepresence of specific antigenic polysaccharides. So far, 35 differentserotypes have been described (9, 56, 14). In several Europeancountries, S. suis serotype 2 is the most prevalent type isolated fromdiseased pigs, followed by serotypes 9 and 1. Serological typing of S.suis is performed using different types of agglutination tests. In thesetests, isolated and biochemically characterized S. suis cells areagglutinated with a panel of 35 specific sera. These methods are verylaborious and time-consuming.

Little is known about the pathogenesis of the disease caused by S. suis,let alone about its various serotypes such as type 2. Various bacterialcomponents, such as extracellular and cell-membrane associated proteins,fimbriae, hemagglutinins, and hemolysis, have been suggested asvirulence factors (9, 10, 11, 15, 16, 47, 49). However, the precise roleof these protein components in the pathogenesis of the disease remainsunclear (37). It is well known that the polysaccharide capsule ofvarious Streptococci and other gram-positive bacteria plays an importantrole in pathogenesis (3, 6, 35, 51, 52). The capsule enables thesemicroorganisms to resist phagocytosis and is therefore regarded as animportant virulence factor. Recently, a role of the capsule of S. suisin the pathogenesis was suggested as well (5). However, the structure,organization and function of the genes responsible for capsulepolysaccharide synthesis (“cps”) in S. suis is unknown. Within S. suis,serotype 1 and 2 strains can differ in virulence for pigs (41, 45, 49).Some type 1 and 2 strains are virulent, other strains are not. Becauseboth virulent and non-virulent strains of serotype 1 and 2 strains arefully encapsulated, it may even be that the capsule is not a relevantfactor required for virulence.

Attempts to control S. suis infections or disease are still hampered bythe lack of knowledge about the epidemiology of the disease and the lackof effective vaccines and sensitive diagnostics. It is well known andgenerally accepted that the polysaccharide capsule of variousStreptococci and other gram-positive bacteria plays an important role inpathogenesis. The capsule enables these microorganisms to resistphagocytosis and is therefore regarded as an important virulence factor.

Compared to encapsulated S. suis strains, non-encapsulated S. suisstrains are phagocytosed by murine polymorphonuclear leucocytes to agreater degree. Moreover, an increase in thickness of capsule was notedfor in vivo grown virulent strains while no increase was observed foravirulent strains. Therefore, these data again demonstrate the role ofthe capsule in the pathogenesis for S. suis as well.

Ungrouped Streptoccus species, such as S. mutans, causing caries inhumans, S. uberis, causing mastitis in cattle, and S. pneumonia, causingmajor infections in humans, and Enterococcus faecilalis and E. faecium,further contribute to the large group of Streptococci.

Streptococcus pneumoniae (the pneumococcus) is a human pathogen causinginvasive diseases, such as pneumonia, bacteremia, and meningitis.Despite the availability of antibiotics, pneumococcal infections remaincommon and can still be fatal, especially in high-risk groups, such asyoung children and elderly people. Particularly in developing countries,many children under the age of five years die each year frompneumococcal pneumonia. S. pneumoniae is also the leading cause ofotitis media and sinusitis. These infections are less serious, butnevertheless incur substantial medical costs, especially when leading tocomplications, such as permanent deafness. The normal ecological nicheof the pneumococcus is the nasopharynx of man. The entire humanpopulation is colonized by the pneumococcus at one time or another, andat a given time, up to 60% of individuals may be carriers.Nasopharyngeal carriage of pneumococci by man is often accompanied bythe development of protection against infection by the same serotype.Most infections do not occur after prolonged carriage but followexposure to recently acquired strains. Many bacteria contain surfacepolysaccharides that act as a protective layer against the environment.Surface polysaccharides of pathogenic bacteria usually make the bacteriaresistant to the defense mechanisms of the host, for example, the lyticaction of serum or phagocytosis. In this respect, the serotype-specificcapsular polysaccharide (“CP”) of Streptococcus pneumoniae, is animportant virulence factor. Unencapsulated strains are avirulent, andantibodies directed against the CP are protective. Protection isserotype specific; each serotype has its own, specific CP structure.Ninety different capsular serotypes have been identified. Currently, CPsof 23 serotypes are included in a vaccine.

Vaccines directed against Streptococcus infections typically aim toutilize an immune response directed against the polysaccharide capsuleof the various Streptococcus species, especially since the capsule isconsidered a primary virulence factor for these bacteria. Duringinfection, the capsule provides resistance against phagocytosis and thusprotects the bacteria from the immune system of the host, and fromelimination by macrophages and neutrophils.

The capsule particularly confers the bacterium resistance tocomplement-mediated opsonophagocytosis. In addition, some bacteriaexpress capsular polysaccharides (CPs) that mimic host molecules,thereby avoiding the immune system of the host. Also, even when thebacteria have been phagocytosed, intracellular killing is hampered bythe presence of a capsule.

It is generally thought that the bacterium will get recognized by theimmune system through the anticapsular-antibodies or serum-factors boundto its capsule and will, through opsonization, get phagocytosed andkilled only when the host has antibodies or other serum factors directedagainst capsule antigens.

However, these antibodies are serotype-specific, and will often onlyconfer protection against only one of the many serotypes known within agroup of Streptococci.

For example, current commercially available S. suis vaccines, which aregenerally based on whole-cell-bacterial preparations, or oncapsule-enriched fractions of S. suis, confer only limited protectionagainst heterologous strains. Also, the current pneumococcal vaccinethat was licensed in the United States in 1983, consists of purified CPsof 23 pneumococcal serotypes whereas at least 90 CP types exist.

The composition of this pneumococcal vaccine was based, on the frequencyof the occurrence of disease isolates in the US and cross-reactivitybetween various serotypes. Although this vaccine protects healthy adultsagainst infections caused by serotypes included in the vaccine, it failsto raise a protective immune response in infants younger than 18 monthsand it is less effective in elderly people. In addition, the vaccineconfers only limited protection in patients with immunodeficiencies andhematology malignancies.

DISCLOSURE OF THE INVENTION

Provided is an isolated or recombinant nucleic acid encoding a capsular(cps) gene cluster of Streptococcus suis. Biosynthesis of capsulepolysaccharides has generally been studied in a number of Gram-positiveand Gram-negative bacteria (32). In Gram-negative bacteria, but also ina number of Gram-positive bacteria, genes which are involved in thebiosynthesis of polysaccharides are clustered at a single locus.

Streptococcus suis capsular genes, as provided herein, show a commongenetic organization involving three distinct regions. The centralregion is serotype specific and encodes enzymes responsible for thesynthesis and polymerization of the polysaccharides. The central regionis flanked by two regions conserved in S. suis that encode proteins forcommon functions, such as transport of the polysaccharide across thecellular membrane. However, between species, only low homologies exist,hampering easy comparison and detection of seemingly similar genes.Knowing the nucleic acid encoding the flanking regions allowstype-specific determination of nucleic acid of the central region ofStreptococcus suis serotypes as, for example, described herein.

Provided is an isolated or recombinant nucleic acid encoding a capsulargene cluster of Streptococcus suis or a gene or gene fragment derivedtherefrom. Such a nucleic acid is, for example, provided by hybridizingchromosomal DNA derived from any one of the Streptococcus suis serotypesto a nucleic acid encoding a gene derived from a Streptococcus suisserotype 1, 2 or 9 capsular gene cluster, as provided herein (see, forexample, Tables 4 and 5) and cloning of (type-specific) genes as, forexample, described herein. At least 14 open reading frames areidentified. Most of the genes belong to a single transcriptional unit,identifying a coordinate control of these genes. The genes, and theenzymes and proteins they encode, act in concert to provide the capsulewith the relevant polysaccharides.

Provided is cps genes and proteins encoded thereof involved inregulation (CpsA), chain length determination (CpsB, C), export (CpsC)and biosynthesis (CpsE, F, G, H, J, K). Although, at first glance, theoverall organization seemed to be similar to that of the cps and epsgene clusters of a number of Gram-positive bacteria (19, 32, 42),overall homologies are low (see, table 3). The region involved inbiosynthesis is located at the center of the gene cluster and is flankedby two regions containing genes with more common functions.

Provided is an isolated or recombinant nucleic acid encoding a capsulargene cluster of Streptococcus suis serotype 2, or a gene or genefragment derived therefrom, preferably as identified in FIG. 3. Genes inthis gene cluster are involved in polysaccharide biosynthesis ofcapsular components and antigens. For a further description of suchgenes see, for example, Table 2. For example, a cpsA gene is providedfunctionally encoding regulation of capsular polysaccharide synthesis,whereas cpsB and cpsC are functionally involved in chain-in-chain lengthdetermination. Other genes, such as cpsD, E, F, G, H, I, J, K andrelated genes, are involved in polysaccharide synthesis, functioning,for example, as glucosyl- or glycosyltransferase. The cpsF, G, H, I, Jgenes encode more type-specific proteins than the flanking genes whichare found more-or-less conserved throughout the species and can serve asa base for selection of primers or probes in PCR-amplification orcross-hybridization experiments for subsequent cloning.

Further provided is an isolated or recombinant nucleic acid encoding acapsular gene cluster of Streptococcus suis serotype 1 or a gene or genefragment derived therefrom, preferably as identified in FIG. 4.

In addition, provided is an isolated or recombinant nucleic acidencoding a capsular gene cluster of Streptococcus suis serotype 9 or agene or gene fragment derived therefrom, preferably as identified inFIG. 5.

Furthermore, provided is, for example, a fragment of the cps locus, orparts thereof, involved in the capsular polysaccharide biosynthesis ofS. suis exemplified herein for serotypes 1, 2 or 9, and allows easyidentification or detection of related fragments derived of otherserotypes of S. suis.

Provided is a nucleic acid probe or primer derived from a nucleic acidaccording to the invention allowing species or serotype-specificdetection of Streptococcus suis. Such a probe or primer (usedinterchangeably herein) is, for example, a DNA, RNA or PNA (peptidenucleic acid) probe hybridizing with capsular nucleic acid as providedherein. Species-specific detection is provided preferably by selecting aprobe or primer sequence from a species-specific region (e.g., flankingregion) whereas serotype-specific detection is provided preferably byselecting a probe or primer sequence from a type-specific region (e.g.,central region) of a capsular gene cluster as provided herein. Such aprobe or primer can be used in a further unmodified form, for example,in cross-hybridization or polymerase-chain reaction (PCR) experimentsas, for example described in the experimental part herein. Provided isthe isolation and molecular characterization of additional type-specificcps genes of S. suis types 1 and 9. In addition, we describe the geneticdiversity of the cps loci of serotypes 1, 2 and 9 among the 35 S. suisserotypes known. Type-specific probes are identified. Also, atype-specific PCR, for example, for serotype 9, is provided, being arapid, reliable and sensitive assay used directly on nasal or tonsillarswabs or other samples of infected or carrier animals.

Also provided is a probe or primer with at least one reporter molecule.Examples of reporter molecules are manifold and known in the art; forexample, a reporter molecule can include additional nucleic acidprovided with a specific sequence (e.g., oligo-dT) hybridizing to acorresponding sequence in which hybridization can easily be detected,for example, because it has been immobilized to a solid support.

Yet other reporter molecules include chromophores, e.g., fluorochromesfor visual detection, for example, by light microscopy or fluorescent insitu hybridization (“FISH”) techniques, or include an enzyme such ashorseradish peroxidase for enzymatic detection, e.g., in enzyme-linkedassays (“EIA”). Yet other reporter molecules include radioactivecompounds for detection in radiation-based assays.

In certain embodiments, at least one probe or primer according to theinvention is provided (labeled) with a reporter molecule and a quenchermolecule, together with an unlabeled probe or primer in a PCR-based testallowing rapid detection of specific hybridization.

Further provided is a diagnostic test or test kit including a probe orprimer as provided herein. Such a test or test kit is, for example, across-hybridization test or PCR-based test advantageously used in rapiddetection and/or serotyping of Streptococcus suis.

Further provided is a protein or fragment thereof encoded by a nucleicacid according to the invention. Examples of such a protein or fragmentare proteins described in Table 2. For example, a cpsA protein isprovided that functionally encodes regulation of capsular polysaccharidesynthesis, whereas cpsB and cpsC are functionally involved inchain-in-chain length determination. Other proteins or functionalfragments thereof, as provided herein, such as cpsD, E, F, G, H, I, J, Kand related proteins, are involved in polysaccharide biosynthesis,functioning, for example, as glucosyl- or glycosyltransferase inpolysaccharide biosynthesis of Streptococcus suis capsular antigen.

Also provided is a method of producing a Streptococcus suis capsularantigen including using a protein or functional fragment thereof asprovided herein, and provides therewith a Streptococcus suis capsularantigen obtainable by such a method.

A comparison of the predicted amino acid sequences of the cps2 geneswith sequences found in the databases allowed the assignment offunctions to the open reading frames. The central region contains thetype-specific glycosyltransferases and the putative polysaccharidepolymerase. This region is flanked by two regions encoding for proteinswith common functions, such as regulation and transport ofpolysaccharide across the membrane. Biosynthesis of Streptococcuscapsular polysaccharide antigen using a protein or functional fragmentthereof is advantageously used in chemo-enzymatic synthesis and thedevelopment of vaccines which offer protection against serotype-specificStreptococcal disease, and is also advantageously used in the synthesisand development of multivalent vaccines against Streptococcalinfections. Such vaccines elicit anticapsular antibodies which conferprotection.

Furthermore, provided is an acapsular Streptococcus mutant for use in avaccine, a vaccine strain derived therefrom and a vaccine derivedtherefrom. Surprisingly, and against the grain of common doctrine,provided is use of a Streptococcus mutant deficient in capsularexpression in a vaccine.

Acapsular Streptococcus mutants have long been known in the art and canbe found in nature. Griffith (J. Hyg. 27:113-159, 1928) demonstratedthat pneumococci could be transformed from one type to another. If heinjected live rough (acapsular or unencapsulated) type 2 pneumococciinto mice, the mice would survive. If, however, he injected the samedose of live rough type 2 mixed with heat-killed smooth (encapsulated)type 1 into a mouse, the mouse would die, and, from the blood, he couldisolate live smooth type 1 pneumococci. At that time, the significanceof this transforming principle was not understood. However,understanding came when it was shown that DNA constituted the geneticmaterial responsible for phenotypic changes during transformation.

Streptococcus mutants deficient in capsular expression are found inseveral forms. Some are fully deficient and have no capsule at all,others form a deficient capsule, characterized by a mutation in acapsular gene cluster. Deficiency can, for instance, include capsularformation wherein the organization of the capsular material has beenrearranged as, for example, demonstrable by electron microscopy. Yetothers have a nearly fully developed capsule which is only deficient ina particular sugar component.

Now, after much advance of biotechnology and despite the fact thatlittle is still known about the exact localization and sequence of genesinvolved in capsular synthesis in Streptococci, it is possible to createmutants of Streptococci, for example, by homologous recombination ortransposon mutagenesis, which has, for example, been done for GAS(Wessels et al., PNAS 88:8317-8321, 1991), for GBS (Wessels et al., PNAS86: 8983-8987, 1989), for S. suis (Smith, ID-DLO Annual report 1996,page 18-19; Charland et al., Microbiol. 144:325-332, 1998) and S.pneumoniae (Kolkman et al., J. Bact. 178:3736-3741, 1996). Suchrecombinant derived mutants, or isogenic mutants, can easily be comparedwith the wild-type strains from which they have been derived.

In certain embodiments, provided is use of a recombinant-derivedStreptococcus mutant deficient in capsular expression in a vaccine.Recombinant techniques useful in producing such mutants are, forexample, homologous recombination, transposon mutagenesis, and others,wherein deletions, insertions or (point) mutations are introduced in thegenome. Advantages of using recombinant techniques include the stabilityof the obtained mutants (especially with homologous recombination anddouble cross-over techniques), and the knowledge about the exact site ofthe deletion, mutation or insertion.

In another embodiment, provided is a stable mutant deficient in capsularexpression obtained, for example, through homologous recombination orcross-over integration events. Examples of such a mutant can be foundherein, such as mutants 10cpsB or 10cpsEF are stable mutants as providedherein.

Also provided is a Streptococcus vaccine strain and vaccine that hasbeen derived from a Streptococcus mutant deficient in capsularexpression. In general, the strain or vaccine is applicable within thewhole range of Streptococcal infections, including animals or man orwith zoonotic infections. It is, of course, now possible to first selecta common vaccine strain and derive a Streptococcus mutant deficient incapsular expression thereof for the selection of a vaccine strain anduse in a vaccine according to the invention.

In certain embodiments, provided is use of a Streptococcus mutantdeficient in capsular expression in a vaccine wherein the Streptococcusmutant is selected from the group composed of Streptococcus group A,Streptococcus group B, Streptococcus suis and Streptococcus pneumoniae.Herewith provided is vaccine strains and vaccines for use with thesenotoriously heterologous Streptococci, of which a multitude of serotypesexist. With a vaccine as provided by herein, which is derived from aspecific Streptococcus mutant deficient in capsular expression, thedifficulties relating to lack of heterologous protection can becircumvented since these mutants do not rely on capsular antigens, perse, to induce protection.

In certain embodiments, the vaccine strain is selected for its abilityto survive, or even replicate, in an immune-competent host or host cellsand thus can persist for a certain period, varying from 1-2 days to morethan one or two weeks, in a host, despite its deficient character.

Although an immunodeficient host will support replication of a widerange of bacteria that are deficient in one or more virulence factors,in general, it is considered a characteristic of pathogenicity ofStreptococci that they can survive for certain periods or replicate in anormal host or host cells such as macrophages. For example, Williams andBlakemore (Neuropath. Appl. Neurobiol. 16, 345-356, 1990; Neuropath.Appl. Neurobiol. 16, 377-392, 1990; J. Infect. Dis. 162, 474-481, 1990)show that both polymorphonuclear cells and macrophage cells are capableof phagocytosing pathogenic S. suis in pigs lacking anti-S. suisantibodies; only pathogenic bacteria could survive and multiply insidemacrophages and the pig.

In certain embodiments, the invention, however, provides a deficient oravirulent mutant or vaccine strain which is capable of surviving atleast 4-5 days, preferably at least 8-10 days in the host, therebyallowing the development of a solid immune response to subsequentStreptococcus infection,

Due to its persistent but avirulent character, a Streptococcus mutant orvaccine strain, as provided herein, is well suited to generate specificand/or long-lasting immune responses against Streptococcal antigens.Moreover, possible specific immune responses of the host directedagainst a capsule are relatively irrelevant because a vaccine strain, asprovided herein, is typically not recognized by such antibodies.

In addition, provided is a Streptococcus vaccine strain, which strainincludes a mutant capable of expressing a Streptococcus virulence factoror antigenic determinant.

In certain embodiments, provided is a Streptococcus vaccine strain,which includes a mutant capable of expressing a Streptococcus virulencefactor wherein the virulence factor or antigenic determinant is selectedfrom a group of cellular components, such as muramidase-released protein(“MRP”), extracellular factor (“EF”), and cell-membrane associatedproteins, 60 kDA heat shock protein, pneumococcal surface protein A (PspA), pneumolysin, C protein, protein M, fimbriae, hemagglutinins andhemolysis or components functionally related thereto.

In certain embodiments, provided is a Streptococcus vaccine strainincluding a mutant capable of over-expressing the virulence factor. Inthis way, provided is a vaccine strain for incorporation in a vaccinewhich specifically causes a host immune response directed againstantigenically important determinants of virulence (listed above),thereby providing specific protection against the determinants.Over-expression can, for example, be achieved by cloning the geneinvolved behind a strong promoter, which is, for example,constitutionally expressed in a multicopy system, either in a plasmid orvia integration in a genome.

In yet another embodiment, provided is a Streptococcus vaccine strain,including a mutant capable of expressing a non-Streptococcus protein.Such a vector-Streptococcus vaccine strain allows, when used in avaccine, protection against pathogens other than Streptococcus.

Due to its persistent but avirulent character, a Streptococcus vaccinestrain or mutant as provided herein is well suited to generate specificand long-lasting immune responses, not only against Streptococcalantigens, but also against other antigens expressed by the strain.Specifically, antigens derived from another pathogen are now expressedwithout the detrimental effects of the antigen or pathogen which wouldotherwise have harmed the host.

An example of such a vector is a Streptococcus vaccine strain or mutantwherein the antigen is derived from a pathogen, such as Actinobacilluspleuropneumonia, oplasmatae, Bordetella, pasteurella, E. coli,Salmonella, campylobacter, Serpulina and others.

Also provided is a vaccine including a Streptococcus vaccine strain ormutant and a pharmaceutically acceptable carrier or adjuvant. Carriersor adjuvants are well known in the art; examples are phosphate bufferedsaline, physiological salt solutions, (double-) oil-in-water emulsions,aluminumhydroxide, Specol, block- or co-polymers, and others.

A vaccine according to the invention can include a vaccine strain eitherin a killed or live form. For example, a killed vaccine including astrain having (over)expressed a Streptococcal or heterologous antigen orvirulence factor is very well suited for eliciting an immune response.In certain embodiments, provided is a vaccine wherein the strain islive, due to its persistent but avirulent character; a Streptococcusvaccine strain, as provided herein, is well suited to generate specificand long-lasting immune responses.

Also provided is a method for controlling or eradicating a Streptococcaldisease in a population, the method comprising vaccinating subjects inthe population with a vaccine according to the invention.

In certain embodiments, a method for controlling or eradicating aStreptococcal disease is provided including testing a sample, such as ablood sample, or nasal or throat swab, feces, urine, or other samplessuch as can be sampled at or after slaughter, collected from atleast-one subject, such as an infant or a pig, in a population partly orwholly vaccinated with a vaccine according to the invention for thepresence of encapsulated Streptococcal strains or mutants. Since avaccine strain or mutant according to the invention is not pathogenic,and can be distinguished from wild-type strains by capsular expression,the detection of (fully) encapsulated Streptococcal strains indicatesthat wild-type infections are still present. Such wild-type infectedsubjects can then be isolated from the remainder of the population untilthe infection has passed. With domestic animals, such as pigs, it iseven possible to remove the infected subject from the population as awhole by culling. Detection of wild-type strains can be achieved viatraditional culturing techniques, or by rapid detection techniques suchas PCR detection.

In yet another embodiment, provided is a method for controlling oreradicating a Streptococcal disease including testing a sample collectedfrom at least one subject in a population partly or wholly vaccinatedwith a vaccine according to the invention for the presence ofcapsule-specific antibodies directed against Streptococcal strains.Capsule-specific antibodies can be detected with classical techniquesknown in the art, such as used for Lancefield's group typing orserotyping.

One embodiment for controlling or eradicating a Streptococcal disease ina population includes vaccinating subjects in the population with avaccine according to the invention and testing a sample collected fromat least one subject in the population for the presence of encapsulatedStreptococcal strains and/or for the presence of capsule-specificantibodies directed against Streptococcal strains.

For example, a method is provided wherein the Streptococcal disease iscaused by Streptococcus suis.

Also provided is a diagnostic assay for testing a sample for use in amethod according to the invention including at least one means for thedetection of encapsulated Streptococcal strains and/or for the detectionof capsule-specific antibodies directed against Streptococcal strains.

Further provided is a vaccine including an antigen according to theinvention and a suitable carrier or adjuvant. The immunogenicity of acapsular antigen provided herein is, for example, increased by linkingto a carrier (such as a carrier protein), allowing the recruitment ofT-cell help in developing an immune response.

Further provided is a recombinant microorganism provided with at least apart of a capsular gene cluster derived from Streptococcus suis.Provided is for example a lactic acid bacterium provided with at least apart of a capsular gene cluster derived from Streptococcus suis. Variousfood-grade lactic acid bacteria (Lactococcus lactis, Lactobacilluscasei, Lactobacillus plantarium and Streptococcus gordonii) have beenused as delivery systems for mucosal immunization. It has now been shownthat oral (or mucosal) administration of recombinant L. lactis,Lactobacillus, and Streptococcus gordonii can elicit local IgA and/orIgG antibody responses to an expressed antigen. The use of oral routesfor immunization against infective diseases is desirable because oralvaccines are easier to administer and have higher compliance rates, andbecause mucosal surfaces are the portals of entry for many pathogenicmicrobial agents. It is within the skill of the artisan to provide suchmicroorganisms with (additional) genes.

Further provided is a recombinant Streptococcus suis mutant providedwith a modified capsular gene cluster. It is within the skill of theartisan to swap genes within a species. In certain embodiments, anavirulent Streptococcus suis mutant is selected to be provided with atleast a part of a modified capsular gene cluster according to theinvention.

Further provided is a vaccine including a microorganism or a mutantprovided herein. An advantage of such a vaccine over currently usedvaccines is that they include accurately defined microorganisms andwell-characterized antigens, allowing accurate determination of immuneresponses against various antigens of choice.

The invention is further explained in the experimental part of thisdescription without limiting the invention thereto.

DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the organization of the cps2 gene cluster of S. suistype 2.

(A) Genetic map of the cps2 gene cluster. The shadowed arrows representpotential ORFs. Interrupted ORFs indicate the presence of stop codons orframe-shift mutations. Gene designations are indicated below the ORFs.The closed arrows indicate the position of the potential promotersequences. | indicates the position of the potential transcriptionregulator sequence. |∥ indicates the position of the 100-bp repeatedsequence.

(B) Physical map of the cps2 locus. Restriction sites are as follows: A:AluI; C: ClaI; E: EcoRI; H: HindIII; K: KpnI; M: MluI; N: NsiI; P: PstI;S: SnaBI; Sa: SacI; X: XbaI.

(C) The DNA fragments cloned in the various plasmids.

FIG. 2 illustrates ethidium bromide stained agarose gel showing PCRproducts obtained with chromosomal DNA of S. suis strains belonging tothe serotypes 1, 2, ½, 9 and 14 and cps2J, cps1I, and cps9H primer setsas described herein.

(A) cps1I primers; (B) cps2J primers and (C) cps9H primers.

Lanes 1-3: serotype 1 strains; lanes 4-6: serotype 2 strains; lanes 7-9:serotype ½ strains; lanes 10-12: serotype 9 strains and lanes 13-15:serotype 14 strains.

(B) Ethidium bromide stained agarose gel showing PCR products obtainedwith tonsillar swabs collected from pigs carrying S. suis type 2, type 1or type 9 strains and cps2J, cps1I and cpsH primer sets as described inMaterials and Methods. Bacterial DNA suitable for PCR was prepared byusing the multiscreen methods as described previously (20).

(C) cps1I primers. (B) cps2J primers and (C) cps9H primers.

Lanes 1-3: PCR products obtained with tonsillar swabs collected frompigs carrying S. suis type 1 strains; lanes 4-6: PCR products obtainedwith tonsillar swabs collected from pigs carrying S. suis type 2strains; lanes 7-9: PCR products obtained with tonsillar swabs collectedfrom pigs carrying S. suis type 9 strains; lanes 10-12: PCR productsobtained with chromosomal DNA from serotype 9, 2 and 1 strainsrespectively; lane 13: negative control, no DNA present.

FIG. 3 illustrates the CPS2 nucleotide sequences and corresponding aminoacid sequences from the open reading frames.

FIG. 4 illustrates the CPS1 nucleotide sequences and corresponding aminoacid sequences from the open reading frames.

FIG. 5 illustrates the CPS9 nucleotide sequences and corresponding aminoacid sequences from the open reading frames.

FIG. 6 illustrates the CPS7 nucleotide sequences and corresponding aminoacid sequences from the open reading frames.

FIG. 7 illustrates alignment of the N-terminal parts of Cps2J and Cps2K.

Identical amino acids are marked by bars. The amino acids shown in boldare also conserved in Cps14I, Cps24J of S. pneumoniae and several otherglycosyltransferases (19). The aspartate residues marked by asterisksare strongly conserved.

FIG. 8 illustrates transmission electron micrographs of thin sections ofvarious S. suis strains.

(A) wild-type strain 10;

(B) mutant strain 10cpsB;

(C) mutant strain 10cpsEF.

Bar=100 nm

FIG. 9 illustrates the kinetics of phagocytosis of wild-type and mutantS. suis strains.

(A) Kinetics of phagocytosis of wild-type and mutant S. suis strains byporcine alveolar macrophages. Phagocytosis was determined as describedherein. The Y-axis represents the number of CFU per milliliter in thesupernatant fluids as determined by plate counting, the X-axisrepresents time in minutes.

□ wild-type strain 10;

∘ mutant strain 10cpsB;

Δ mutant strain 10cpsEF.

(B) Kinetics of intracellular killing of wild-type and mutant S. suisstrains by porcine AM. The intracellular killing was determined asdescribed herein. The Y-axis represents the number of CFU per ml in thesupernatant fluids after lysis of the macrophages as determined by platecounting, the X-axis represents time in minutes.

□ wild-type strain 10;

∘ mutant strain 10cpsB;

Δ mutant strain 10cpsEF.

FIG. 10 illustrates the nucleotide sequence alignment of the highlyconserved 100-bp repeated element.

1) 100-bp repeat between cps2G and cps2H

2) 100-bp repeat within “cps2M”

3) 100-bp repeat between cps20 and cps2P

FIG. 11 illustrates the cps2, cps9 and cps7 gene clusters of S. suisserotypes 2, 9 and 7.

(A) Genetic organization of the cps2 gene cluster [84]. The large arrowsrepresent potential ORFs. Gene designations are indicated below theORFs. Identically filled arrows represent ORFs which showed homology.The small closed arrows indicate the position of the potential promotersequences. | indicates the position of the potential transcriptionregulator sequence.

(B) Physical map and genetic organization of the cps9 gene cluster [15].Restriction sites are as follows: B: BamHI; P: PstI; H: HindIII; X:XbaI. The DNA fragments cloned in the various plasmids are indicated.The open arrows represent potential ORFs.

(C) Physical map and genetic organization of the cps7 gene cluster.Restriction sites are as follows: C: ClaI; P: PstI; Sc: ScaI. The DNAfragments cloned in the various plasmids are indicated. The open arrowsrepresent potential ORFs.

FIG. 12 illustrates ethidium bromide stained agarose gel showing PCRproducts.

(A) Ethidium bromide stained agarose gel showing PCR products obtainedwith chromosomal DNA of S. suis strains belonging to the serotypes 1, 2,9 and 7 and the cps7H primer set. Strain designations are indicatedabove the lanes. C: negative control, no DNA present. M: molecular sizemarker (lambda digested with EcoRI and HindIII).

(B) Ethidium bromide stained agarose gel showing PCR products obtainedwith serotype 7 strains collected in different countries and fromdifferent organs. Bacterial DNA suitable for PCR was prepared by usingthe multiscreen method as described herein [89]. Strain designations areindicated above the lanes. M: molecular size marker (lambda digestedwith EcoRI and HindIII).

DETAILED DESCRIPTION OF THE INVENTION Experimental Part MATERIAL ANDMETHODS

Bacterial strains and growth conditions. The bacterial strains andplasmids used in this study are listed in Table 1. S. suis strains weregrown in Todd-Hewitt broth (code CM 189, Oxoid), and plated on Columbiaagar blood base (code CM331, Oxoid) containing 6% (v/v) horse blood. E.coli strains were grown in Luria broth (28) and plated on Luria brothcontaining 1.5% (w/v) agar. If required, antibiotics were added to theplates at the following concentrations: spectinomycin: 100 μg/ml for S.suis and 50 μg/ml for E. coli and ampicillin, 50 μg/ml.

Serotyping. The S. suis strains were serotyped by the slideagglutination test with serotype-specific antibodies (44).

DNA techniques. Routine DNA manipulations were performed as described bySambrook et al. (36).

Alkaline phosphatase activity. To screen for PhoA fusions in E. coli,plasmid libraries were constructed. Therefore, chromosomal DNA of S.suis type 2 was digested with AluI. The 300-500-bp fragments wereligated to SmaI-digested pPHOS2. Ligation mixtures were transformed tothe PhoA⁻ E. coli strain CC118. Transformants were plated on LB mediasupplemented with 5-Bromo-4-chloro-3-indolylfosfaat (BCIP, 50 μg/ml,Boehringer, Mannheim, Germany). Blue colonies were purified on freshLB/BCIP plates to verify the blue phenotype.

DNA sequence analysis. DNA sequences were determined on a 373A DNASequencing System (Applied Biosystems, Warrington, GB). Samples wereprepared by using an ABI/PRISM dye terminator cycle sequencing readyreaction kit (Applied Biosystems). Sequencing data were assembled andanalyzed using the MacMollyTetra program. Custom-made sequencing primerswere purchased from Life Technologies. Hydrophobic stretches withinproteins were predicted by the method of Klein et al. (17). The BLASTprogram available on Netscape Navigator™ was used to search for proteinsequences related to the deduced amino acid sequences.

Construction of gene-specific knock-out mutants of S. suis. To constructthe mutant strains 10cpsB and 10cpsEF, we electrotransformed thepathogenic serotype 2 strain 10 (45, 49) of S. suis with pCPS11 andpCPS28 respectively. In these plasmids, the cpsB and cpsEF genes weredisturbed by the insertion of a spectinomycin-resistance gene. To createpCPS11, the internal 400 bp PstI-BamHI fragment of the cpsB gene inpCPS7 was replaced by the Spc^(R) gene. For this purpose, pCPS7 wasdigested with PstI and BamHI and ligated to the 1,200-bp PstI-BamHIfragment, containing the Spc^(R) gene, from pIC-spc. To constructpCPS28, we have used pIC20R. In this plasmid we inserted the KpnI-SalIfragment from pCPS17 (resulting in pCPS25) and the XbaI-ClaI fragmentfrom pCPS20 (resulting in pCPS27). pCPS27 was digested with PstI andXhoI and ligated to the 1,200-bp PstI-XhoI fragment, containing theSpc^(R) gene of pIC-spc. The electrotransformation to S. suis wascarried out as described before (38).

Southern blotting and hybridization. Chromosomal DNA was isolated asdescribed by Sambrook et al. (36). DNA fragments were separated on 0.8%agarose gels and transferred to Zeta-Probe GT membranes (Bio-Rad) asdescribed by Sambrook et al. (36). DNA probes were labeled with[(-³²P]dCTP (3000 Ci mmol⁻¹ Amersham) by use of a random primed labelingkit (Boehringer). The DNA on the blots was hybridized at 65° C. withappropriate DNA probes as recommended by the supplier of the Zeta-Probemembranes. After hybridization, the membranes were washed twice with asolution of 40 mM sodium phosphate, pH 7.2, 1 mM EDTA, 5% SDS for 30 minat 65° C. and twice with a solution of 40 mM sodium phosphate, pH 7.2, 1mM EDTA, 1% SDS for 30 min at 65° C.

PCR. The primers used in the cps2J PCR correspond to the positions13791-13813 and 14465-14443 in the S. suis cps2 locus. The sequenceswere: 5′-CAAACGCAAGGAATTACGGTATC-3′ (SEQ ID NO:1) and5′-GAGTATCTAAAGAATGCCTATTG-3′ (SEQ ID NO:2). The primers used for thecps1I PCR correspond to the positions 4398-4417 and 4839-4821 in the S.suis cps1 sequence. The sequences were: 5′-GGCGGTCTAGCAGATGCTCG-3′ (SEQID NO:3) and 5′-GCGAACTGTTAGCAATGAC-3′ (SEQ ID NO:4). The primers usedin the cps9H PCR correspond to the positions 4406-4126 and 4494-4475 inthe S. suis cps9 sequence. The sequences were:5′-GGCTACATATAATGGAAGCCC3′ (SEQ ID NO:5) and 5′-CGGAAGTATCTGGGCTACTG-3′(SEQ ID NO:6).

Construction of gene-specific knock-out mutants of S. suis. To constructthe mutant strains 10cpsB and 10cpsEF, we electrotransformed thepathogenic serotype 2 strain 10 of S. suis with pCPS11 and pCPS28respectively. In these plasmids, the cpsB and cpsEF genes were disturbedby the insertion of a spectinomycin-resistance gene. To create pCPS11,the internal 400 bp PStI-BamHI fragment of the cpsB gene in pCPS7 wasreplaced by the Spc^(R) gene. For this purpose, pCPS7 was digested withPStI and BamHI and ligated to the 1,200-bp PstI-BamHI fragment,containing the Spc^(R) gene, from pIC-spc. To construct pCPS28, we haveused pIC20R. In this plasmid, we inserted the KpnI-SalI fragment frompCPS17 (resulting in pCPS25) and the XbaI-ClaI fragment from pCPS20(resulting in pCPS27). pCPS27 was digested with PstI and XhoI andligated to the 1,200-bp PstI-XhoI fragment, containing the spc^(R) geneof pIC-Spc. The electrotransformation to S. suis was carried out asdescribed before (38).

Phagocytosis assay. Phagocytosis assays were performed as described byLeij et al. (23). Briefly, to opsonize the cells, 10⁷ S. suis cells wereincubated with 6% SPF-pig serum for 30 min at 37° C. in a head-over-headrotor at 6 rpm. 10⁷ AM and 10⁷ opsonized S. suis cells were combined andincubated at 37° C. under continuous rotation at 6 rpm. At 0, 30, 60 and90 min, 1-ml samples were collected and mixed with 4 ml of ice-cold EMEMto stop phagocytosis. Phagocytes were removed by centrifugation for 4min at 110×g and 4° C. The number of colony-forming units, (“CFU”) inthe supernatants was determined. Control experiments were carried outsimultaneously by combining 10⁷ opsonized S. suis cells with EMEM(without AM).

Killing assays. AM (10⁷/ml) and opsonized S. suis cells (10⁷/ml) weremixed 1:1 and incubated for 10 min at 37° C. under continuous rotationat 6 rpm. Ice-cold EMEM was added to stop further phagocytosis andkilling. To remove extracellular S. suis cells, phagocytes were washedtwice (4 min, 110×g, 4° C.) and resuspended in 5 ml EMEM containing 6%SPF serum. The tubes were incubated at 37° C. under rotation at 6 rpm.After 0, 15, 30, 60 and 90 min, samples were collected and mixed withice-cold EMEM to stop further killing. The samples were centrifuged for4 min at 110×g at 4° C. and the phagocytic cells were lysed in EMEMcontaining 1% saponine for 20 min at room temperature. The number of CFUin the suspensions was determined.

Pigs. Germfree pigs, crossbreeds of Great Yorkshire and Dutch Landrace,were obtained from sows by caesarian sections. The surgery was performedin sterile flexible film isolators. Pigs were allotted to groups, eachconsisting of 4 pigs, and were housed in sterile stainless steelincubators.

Experimental infections. Pigs were inoculated intranasally with S. suistype 2 as described before. To predispose the pigs for infection with S.suis, five-day old pigs were inoculated intranasally with about 10⁷ CFUof Bordetella bronchiseptica strain 92932. Two days later, the pigs wereinoculated intranasally with S. suis type 2 (10⁶ CFU). Pigs weremonitored twice daily for clinical signs of disease, such as fever,nervous signs and lameness. Blood samples were collected three times aweek from each pig. White blood cells were counted with a cell counter.To monitor infection with S. suis and B. bronchiseptica and to check forabsence of contaminants, we collected swabs of nasopharynx and fecesdaily. The swabs were plated directly onto Columbia agar containing 6%horse blood. After three weeks, the pigs were killed and examined forpathological changes. Tissue specimens from the central nervous system,serosae, and joints were examined bacteriologically and histologicallyas described herein (45, 49). Colonization of the serosae was scoredpositively when S. suis was isolated from the pericardium, thoracalpleura or the peritoneum. Colonization of the joints was scoredpositively when S. suis was isolated from one or more joints (12 jointsper animal were scored).

Vaccination and challenge. One week old pigs were vaccinatedintravenously with a dosage of 106 cfu of the S. suis strains 10cpsEF or10cpsB. Three weeks later, the pigs were challenged intravenously withthe pathogenic serotype 2 strain 10 (107 cfu). Disease monitoring,hematological, serological and bacteriological examinations as well aspost-mortum examinations were as described before under experimentalinfections.

Electron Microscopy. Bacteria were prepared for electron microscopy asdescribed by Wagenaar et al. (50). Shortly, bacteria were mixed withagarose MP (Boehringer) of 37° C. to a concentration of 0.7%. Themixture was immediately cooled on ice. Upon gelifying, samples were cutinto 1 to 1.5 mm slices and incubated in a fixative containing 0.8%glutaraldehyde and 0.8% osmiumtetraoxide. Subsequently, the samples werefixed and stained with uranyl acetate by microwave stimulation,dehydrated and imbedded in eponaraldite resin. Ultra-thin sections werecounterstained with lead citrate and examined with a Philips CM 10electron microscope at 80 kV. (FIG. 8.)

Isolation of porcine alveolar macrophages (AM). Porcine AM were obtainedfrom the lungs of specific pathogen-free (“SPF”) pigs. Lung lavagesamples were collected as described by van Leengoed et al. (43). Cellswere suspended in EMEM containing 6% (v/v). SPF-pig serum and adjustedto 10 cells per ml.

RESULTS

Identification of the cps locus. The cps locus of S. suis type 2 wasidentified through a strategy developed for the genetic identificationof exported proteins (13, 31). In this system, a plasmid (pPHOS2)containing a truncated alkaline phosphatase gene (13) was used. The genelacked the promoter sequence, the translational start site and thesignal sequence. The truncated gene is preceded by a unique SmaIrestriction site. Chromosomal DNA of S. suis type 2, digested with AluI,was randomly cloned in this restriction site. Because translocation ofPhoA across the cytoplasmic membrane of E. coli is required forenzymatic activity, the system can be used to select for S. suisfragments containing a promoter sequence, a translational start site anda functional signal sequence. Among 560 individual E. coli clonestested, 16 displayed a dark blue phenotype when plated on mediacontaining BCIP. DNA sequence analysis of the inserts from several ofthese plasmids was performed (results not shown) and the deduced aminoacid sequences were analyzed. The hydrophobicity profile of one of theclones (pPHOS7, results not shown) showed that the N-terminal part ofthe sequence resembled the characteristics of a typical signal peptide:a short hydrophilic N-terminal region is followed by a hydrophobicregion of 38 amino acids. These data indicate that the phoA system wassuccessfully used for the selection of S. suis genes encoding exportedproteins. Moreover, the sequences were analyzed for similarities presentin the databases. The sequence of pPHOS7 showed a high similarity (37%identity) with the protein encoded by the cps14C gene of Streptococcuspneumoniae (19). This strongly suggests that pPHOS7 contains a part ofthe cps operon of S. suis type 2.

Cloning of the flanking cpa genes. In order to clone the flanking cpsgenes of S. suis type 2, the insert of pPHOS7 was used as a probe toidentify chromosomal DNA fragments which contain flanking cps genes. A6-kb HindIII fragment was identified and cloned in pKUN19. This yieldedclone pCPS6 (FIG. 1, part C). Sequence analysis of the insert of pCPS6revealed that pCPS6 most probably contained the 5′-end of the cps locus,but still lacked the 3′-end. Therefore, sequences of the 3′-end of pCPS6were in turn used as a probe to identify chromosomal fragmentscontaining cps sequences located further downstream. These fragmentswere also cloned in pKUN19, resulting in pCPS17. Using the same systemof chromosomal walking, plasmids pCPS18, pCPS20, pCPS23 and pCPS26,containing downstream cps sequences were subsequently generated.

Analysis of the cps operon. The complete nucleotide sequence of thecloned fragments was determined (FIG. 4). Examination of the compiledsequence revealed the presence of at least 13 potential open readingframes (Orfs), which were designated as Orf 2Y, Orf2X and Cps2A-Cps2K(FIG. 1, part A; FIG. 11, part A). Moreover, a 14th, incomplete Orf (Orf2Z) was located at the 5′-end of the sequence. Two potential promotersequences were identified. One was located 313 bp (locations 1885-1865and 1884-1889) upstream of Orf2X. The other potential promoter sequencewas located 68 bp upstream of Orf2Y (locations 2241-2236 and 2216-2211).Orf2Y is expressed in opposite orientation. Between Orfs 2Y and 2Z, thesequence contained a potential stein-loop structure, which could act asa transcription terminator. Each Orf is preceded by a ribosome-bindingsite and the majority of the Orfs are very closely linked. The onlysignificant intergenic gap was found between Cps2G and Cps2H (389nucleotides). However, no obvious promoter sequences or potentialstem-loop structures were found in this region. These data suggest thatOrf2X and Cps2A-Cps2K are arranged as an operon.

An overview of all Orfs with their properties is shown in Table 2. Themajority of the predicted gene products is related to proteins involvedin polysaccharide biosynthesis. Orf2Z showed some similarity with theYitS protein of Bacillus subtilis. YitS was identified during thesequence analysis of the complete genome of B. subtilis. The function ofthe protein is unknown.

Orf2Y showed similarity with the YcxD protein of B. subtilis (53). Basedon the similarity between YcxD and MocR of Rhizobium meliloti (33), YcxDwas suggested to be a regulatory protein.

Orf2X showed similarity with the hypothetical YAAA proteins ofHaemophilus influenzae and E. coli. The function of these proteins isunknown.

The gene products encoded by the cps2A, cps2B, cps2C and cps2D genesshowed approximate similarity to the CpsA, CpsC, CpsD and CpsB proteinsof several serotypes of Streptococcus pneumoniae (19), respectively.This suggests similar functions for these proteins. Hence, Cps2A mayhave a role in the regulation of the capsular polysaccharide synthesis.Cps2B and Cps2C could be involved in the chain length determination ofthe type 2 capsule and Cps2C can play an additional role in the exportof the polysaccharide. The Cps2D protein of S. suis is related to theCpsB protein of S. pneumoniae and to proteins encoded by genes ofseveral other Gram-positive bacteria involved in polysaccharide orexopolysaccharide synthesis, but their function is unknown (19).

The protein encoded by the cps2E gene showed similarity to severalbacterial proteins with glycosyltransferase activities: Cps14E andCps19fE of S. pneumoniae serotypes 14 and 19F (18, 19, 29), CpsE ofStreptococcus salvarius (X94980) and CpsD of Streptococcus agalactiae(34). Recently, Kolkman et al. (18) showed that Cps14E is aglucosyl-1-phosphate transferase that links glucose to a lipid carrier,the first step in the biosynthesis of the S. pneumoniae type 14repeating unit. Based on these data, a similar function may be fulfilledby Cps2E of S. suis.

The protein encoded by the cps2F gene showed similarity to the proteinencoded by the rfbU gene of Salmonella enteritica (25). This similarityis most pronounced in the C-terminal regions of these proteins. The rfbUgene was shown to encode mannosyltransferase activity (25).

The cps2G gene encoded a protein that showed moderate similarity withthe rfbF gene product of Campylobacter hyoilei (22), the epsF geneproduct of S. thermophilus (40) and the capM gene product of S. aureus(24). On the basis of similarity, the rfbF, epsF and capM genes aresuggested to encode galactosyltransferase activities. Hence, a similarglycosyltransferase activity could be fulfilled by the cps2G geneproduct.

The cps2H gene encodes a protein that is similar to the N-terminalregion of the lgtD gene product of Haemophilus influeflzae (U32768).Moreover, the hydrophobicity plots of Cps2H and LgtD looked very similarin these regions (data not shown). Based on sequence similarity, thelgtD gene product was suggested to have glycosyltransferase activity(U32768).

The gene product encoded by the cps2I gene showed some similarity with aprotein of Actinobacillus actinomycetemcomitans (AB002668). This proteinis part of the gene cluster responsible for the serotype-b-specificantigen of A. actinomycetemcomitans. The function of the protein isunknown.

The gene products encoded by the cps2J and cps2K genes showedsignificant similarities to the Cps14J protein of S. pneumoniae. Thecps14J gene of S. pneumoniae was shown to encode aβ-1,4-galactosyltransferase activity. In S. pneumoniae, CpsJ isresponsible for the addition of the fourth (i.e. last) sugar in thesynthesis of the S. pneumoniae serotype 14 polysaccharide (20). Evensome similarity was found between Cps2J and Cps2K (FIG. 2, 25.5%similarity). This similarity was most pronounced in the N-terminalregions of the proteins (FIG. 7). Recently, two small conserved regionswere identified in the N-terminus of Cps14J and Cps14I and theirhomologues (20). These regions were predicted to be important forcatalytic activity. Both regions, DXS and DXDD (FIG. 2), were also foundin Cps2J and Cps2K.

Distribution of the cps2 genes in other S. suis serotypes. To examinethe relationship between the cps2 genes and cps genes in the other S.suis serotypes, we performed cross-hybridization experiments. DNAfragments of the individual cps2 genes were amplified by PCR, labeledwith ³²P, and used to probe Southern blots of chromosomal DNA of thereference strains of the 35 different S. suis serotypes. Largevariations in the hybridization patterns were observed (Table 4). As apositive control, we used a probe specific for 16S rRNA. The 16S rRNAprobe hybridized with all serotypes tested. However, none of the othergenes tested were common in all serotypes. Based on the geneticorganization of the genes, it was previously suggested that orfX andcpsA-cpsK genes are part of one operon and that the proteins encoded bythese genes are all involved in polysaccharide biosynthesis. OrfY andOrfZ are not a part of this operon, and their role in the polysaccharidebiosynthesis is unclear. Based on sequence similarity data, OrfY may beinvolved in regulation of the cps2 genes. OrfZ is proposed to beunrelated to polysaccharide biosynthesis. Probes specific for the orfZ,orfY, orfX, cpsA, cpsB, cpsC and cpsD genes hybridized with most otherserotypes. This suggests that the proteins encoded by these genes arenot type-specific, but may perform more common functions in biosynthesisof the capsular polysaccharide. This confirms previous data which showedthat the cps2A-cps2D genes showed strong similarity to cps genes ofseveral serotypes of Streptococcus pneumoniae. Based on this similarity,Cps2A is possibly a regulatory protein, whereas Cps2B and Cps2C may playa role in length determination and export of polysaccharide. The cps2Egene hybridized with DNA of serotypes 1, 2, 14 and 1/2. The cps2E geneshowed a strong similarity to the cps14E gene of S. pneumoniae (18).This enzyme was shown to have a glucosyl-1-phosphate activity andcatalyzed the transfer of glucose to a lipid carrier (18). These dataindicate that a glycosyltransferase closely related to Cps14E may beresponsible for the first step in the biosynthesis of polysaccharide inthe S. suis serotypes 1, 2, 14 and 1/2. The cps2F, cps2G, cps2H, cps2Iand cps2J genes hybridized with chromosomal DNA of serotypes 2 and 1/2only. The cps2G gene showed an additional weak hybridization signal withDNA of serotype 34. In agglutination tests, serotype 1/2 showedagglutination with sera specific for serotype 2 as well as with seraspecific for serotype 1. This suggests that serotype 1/2 sharesantigenic determinants with both types 1 and 2. The hybridization dataconfirmed these data. All putative glycosyltransferases present inserotype 2 are also present in serotype 1/2. The cps2K gene showed ahybridization pattern similar to the cps2E gene. Hybridization wasobserved with DNA of serotypes 1, 2, 14 and 1/2. Taken together, thesehybridization data show that the cps2 gene cluster can be divided intothree regions: a central region containing the type-specific genes isflanked by two regions containing common genes for various serotypes.

Cloning of the type-specific cps genes of serotypes 1 and 9. To clonethe type-specific cps genes of S. suis serotype 1, the cps2E gene wasused as a probe to identify chromosomal DNA fragments of type 1 whichcontain flanking cps genes. A 5 kb EcoRV fragment was identified andcloned in pKUN19. This yielded pCPS1-1 (FIG. 1, part B). This fragmentwas in turn used as a probe to identify an overlapping 2.2 kb HindIIIfragment. pKUN19 containing this HindIII fragment was designatedpCPS1-2. The same strategy was followed to identify and clone thetype-specific cps genes of serotype 9. In this case, we used the cps2Dgene as a probe. A 0.8 kb HindIII-XbaI fragment was identified andcloned, yielding pCPS9-1 (FIG. 1, part C). This fragment was in turnused as a probe to identify a 4 kb XbaI fragment. pKUN19 containing this4 kb XbaI fragment was designated pCPS9-2.

Analysis of the cloned cps1 genes. The complete nucleotide sequence ofthe inserts of pCPS1-1 and pCPS1-2 was determined (FIG. 5). Examinationof the sequence revealed the presence of five complete and twoincomplete Orfs (FIG. 1, part B). Each Orf is preceded by aribosome-binding site. In accord with data obtained for the cps2 genesof serotype 2, the majority of the Orfs is very closely linked. The onlysignificant gap (718 bp) was found between Cps1G and Cps1H. No obviouspromoter sequences or potential stem-loop structures could be found inthis region. This suggests that, as in serotype 2, the cps genes inserotype 1 are arranged in an operon.

An overview of the Orfs and their properties is shown in Table 2. Asexpected on the basis of the hybridization data (Table 4), the proteinencoded by the cps1E gene was related to Cps2E of S. suis type 2(identity of 86%). The fragment cloned in pCPS1-1 lacked the codingregion for the first 7 amino acids of the cps1E gene.

The protein encoded by the cps1F and cps1 G genes showed strongsimilarity to the Cps14F and Cps14G proteins of Streptococcus pneumoniaeserotype 14, respectively (20). The function of the Cps14F is notcompletely clear, but it has been suggested that Cps14F has a role inglycosyltransferase activity. The cps14G gene of S. pneumoniae was shownto encode β-1,4-galactosyltransferase activity. In S. pneumoniae type14, this activity is required for the second step in the biosynthesis ofthe oligosaccharide subunit (20). Based on the similarity of the data,similar glycosyltransferase and enhancing activities are suggested forthe cps1G and cps1F genes of S. suis type 1.

The protein encoded by the cps1H gene showed similarity to the Cps14Hprotein of S. pneumoniae (20). Based on sequence similarity, Cps14H wasproposed to be the polysaccharide polymerase (20).

The protein encoded by the cps1I gene showed some similarity with theCps14J protein of S. pneumoniae (19). The cps14J gene was shown toencode a β-1,4-galactosyltransferase activity, responsible for theaddition of the fourth (i.e. last) sugar in the synthesis of the S.pneumoniae serotype 14 polysaccharide.

Between Cps1G and Cps1H, a gap of 718 bp was found. This region revealedthree small Orfs. The three Orfs were expressed in three differentreading frames and were not preceded by potential ribosome bindingsites, nor contained potential start sites. However, the three potentialgene products encoded by this region showed some similarity with threesuccessive regions of the C-terminal part of the EpsK protein ofStreptococcus thermophilus (27% identity, 40). The region related to thefirst 82 amino acids is lacking.

Analysis of the cloned cps9 genes. We also determined the completenucleotide sequence of the inserts of pCPS9-1 and pCPS9-2 (FIG. 6).Examination of the sequence revealed the presence of three complete andtwo incomplete Orfs (FIG. 1, part C). As in serotypes 1 and 2, all Orfsare preceded by a ribosome-binding site and are very closely coupled. Assuggested by the hybridization data (Table 4), the Cps2D and Cps9Dproteins were highly related (Table 2). Based on sequence comparisons,pCPS9-1 lacked the first 27 amino acids of the Cps9D protein.

The protein encoded by the cps9E gene showed some similarity with theCapD protein of Staphylococcus aureus serotype 1 (24). Based on sequencesimilarity data, the Cap1D protein was suggested to be an epimerase or adehydratase involved in the synthesis of N-acetylfructosamine orN-acetylgalactosamine (63).

Cps9F showed some similarity to the CapM proteins of S. aureus serotypes5 and 8 (61, 64, 65). Based on sequence similarity data, Cap5M and Cap8Mare proposed to be glycosyltransferases (63).

The protein encoded by the cps9G gene showed some similarity to aprotein of Actinobacillus actinomycetemcomitans (AB002668_(—)4). Thisprotein is part of a gene cluster responsible for the serotypeb-specific antigens of Actinobacillus actinomycetemcomitans. Thefunction of the protein is unknown.

The protein encoded by the cps9H gene showed some similarity to the rfbBgene of Yersinia enterolitica (68). The RfbB protein was shown to beessential for O-antigen synthesis, but the function of the protein inthe synthesis of the O:3 lipopolysaccharide is unknown.

Serotype 1 and serotype 9-specific cps genes. To determine whether thecloned fragments in pCPS1-1, pCPS1-2, pCPS9-1 and pCPS9-2 contained thetype-specific genes for serotype 1 and 9, respectively,cross-hybridization experiments were performed. DNA fragments of theindividual cps1 and cps9 genes were amplified by PCR, labeled with ³²P,and used to probe Southern blots of chromosomal DNA of the referencestrains of the 35 different S. suis serotypes. The results are shown inTable 5. Based on the data obtained with the cps2E probe (Table 4), thecps1E probe was expected to hybridize with chromosomal DNA of S. suisserotypes 1, 2, 14, 27 and 1/2. The cps1H, cps9E and cps9F probeshybridized with most other serotypes. However, the cps1F and cps1 G andcps1I probes hybridized with chromosomal DNA of serotypes 1 and 14 only.The cps9G and cps9H probes hybridized with serotype 9 only. These datasuggest that the cps9G and cps9H probes are specific for serotype 9 and,therefore, could be useful tools for the development of rapid andsensitive diagnostic tests for S. suis type 9 infections.

Type-specific PCR. So far, the probes were tested on the 35 differentreference strains only. To test the diagnostic value of thetype-specific cps probes further, several other S. suis serotype 1, 2,1/2, 9 and 14 strains were used. Moreover, since a PCR-based methodwould be even more rapid and sensitive than a hybridization test, wetested whether we could use a PCR for the serotyping of the S. suisstrains. The oligonucleotide primer sets were chosen within the cps2J,cps1 I and cps9H genes. Amplified fragments of 675 bp, 380 bp and 390 bpwere expected, respectively. The results show that 675 bp fragments wereamplified on type 2 and 1/2 strains using cps2J primers; 380 bpfragments were amplified on type 1 and 14 strains using cps1 I primersand 390 bp fragments were amplified on type 9 strains using cps9Hprimers.

Construction of mutants impaired in capsule production. To evaluate therole of the capsule of S. suis type 2 in pathogenesis, we constructedtwo isogenic mutants in which capsule production was disturbed. Toconstruct mutant 10cpsB, pCPS11 was used. In this plasmid, a part of thecps2B gene was replaced by the spectinomycin-resistance gene. Toconstruct mutant strain 10cpsEF, the plasmid pCPS28 was used. In pCPS28,the 3′-end of cps2E gene, as well as the 5′-end of cps2F gene, werereplaced by the spectinomycin-resistance gene. pCPS11 and pCPS28 wereused to electrotransform strain 10 of S. suis type 2 andspectinomycin-resistant colonies were selected. Southern blotting andhybridization experiments were used to select double cross-overintegration events (results not shown). To test whether the capsularstructure of the strains 10cpsB and 10cpsEF was disturbed, we used aslide agglutination test using a suspension of the mutant strains inhyperimmune anti-S. suis type 2 serum (44). The results showed that evenin the absence of serotype-specific antisera, the bacteria agglutinated.This indicates that, in the mutant strains, the capsular structure wasdisturbed. To confirm this, thin sections of wild-type and mutantstrains were compared by electron microscopy. The results showed that,compared to the wild-type (FIG. 3, part A), the amount of capsuleproduced by the mutant strains was greatly reduced (FIG. 3, parts B andC). Almost no capsular material could be detected on the surface of themutant strains.

Capsular Mutants are Sensitive to Phagocytosis and Killing by PorcineAlveolar Macrophages (“PAM”).

The capsular mutants were tested for their ability to resistphagocytosis by PAM in the presence of porcine SPF serum. The wild-typestrain 10 seemed to be resistant to phagocytosis under these conditions(FIGS. 9A and 9B). In contrast, the mutant strains were efficientlyingested by macrophages (FIGS. 9A and 9B). After 90 minutes, more than99.7% (strain 10cpsB) and 99.8% (strain 10cpsEF) of the mutant cellswere ingested by the macrophages. Moreover, as shown in FIGS. 9A and 9B,the ingested strains were efficiently killed by the macrophages. 90-98%of all ingested cells were killed within 90 min. No differences could beobserved between wild-type and mutant strains. These data indicate thatthe capsule of S. suis type 2 efficiently protects the organism fromuptake by macrophages in vitro.

Capsular mutants are less virulent for germfree piglets. The virulenceproperties of the wild-type and mutant strains were tested afterexperimental infection of newborn germfree pigs (45, 49). Table 1 showsthat specific and nonspecific signs of disease could be observed in allpigs inoculated with the wild-type strain. Moreover, all pigs inoculatedwith the wild-type strain died during the course of the experiment orwere killed because of serious illness or nervous disorders (Table 3).In contrast, the pigs inoculated with strains 10cpsB and 10cpsEF showedno specific signs of disease and all pigs survived until the end of theexperiment (Table 6). The temperature of the pigs inoculated with thewild-type strain increased 2 days after inoculation and remained highuntil day 5 (Table 3). The temperature of the pigs inoculated with themutant strains sometimes exceeded 40° C., however, we could observesignificant differences in the fever index (i.e., percent ofobservations in an experimental group during which pigs showed fever(>40° C.)) between pigs inoculated with wild-type and mutant strains.All pigs showed increased numbers of polymorphonuclear leucocytes (PMLs)(>10×10⁹ PMLs per liter) (Table 3). However, in pigs inoculated with themutant strains, the percentage of samples with increased numbers of PMLswas considerably lower. S. suis strains and B. bronchiseptica could beisolated from the nasopharynx and feces swab samples of all pigs from 1day post-infection until the end of the experiment (Table 3).Postmortem, the wild-type strain could frequently be isolated from thecentral nervous system (“CNS”), kidney, heart, liver, spleen, serosae,joints and tonsils. Mutant strains could easily be recovered from thetonsils, but were never recovered from the kidney, liver or spleen.Interestingly, low numbers of the mutant strains were isolated from theCNS, the serosae, the joints, the lungs and the heart. Taken together,these data strongly indicated that mutant S. suis strains, impaired incapsule production, are not virulent for young germfree pigs.

We describe the identification and the molecular characterization of thecps locus, involved in the capsular polysaccharide biosynthesis, of S.suis. Most of the genes seemed to belong to a single transcriptionalunit, suggesting a coordinate control of these genes. Functions to mostof the gene products were assigned. Regions involved in regulation(Cps2A), chain length determination (Cps2B, C), export (Cps2C) andbiosynthesis (Cps2E, F, G, H, J, K) were identified. The region involvedin biosynthesis is located at the center of the gene cluster and isflanked by two regions containing genes with more common functions. Theincomplete orf2Z gene was located at the 5′-end of the cloned fragment.Orf2Z showed some similarity with the YitS protein of B. subtilis.However, because the function of the YitS protein is unknown, this didnot give us any information about the possible function of Orf2Z.Because the orf2Z gene is not a part of the cps operon, a role of thisgene in polysaccharide biosynthesis is not expected. The Orf2Y proteinshowed some similarity with the YcxD protein of B. subtilis (53). TheYcxD protein was suggested to be a regulatory protein. Similarly, Orf2Ymay be involved in the regulation of polysaccharide biosynthesis. TheOrf2X protein showed similarity with the YAAA proteins of H. influenzaeand E. coli. The function of these proteins is unknown. In S. suis type2, the orf2X gene seemed to be the first gene in the cps2 operon. Thissuggests a role of Orf2X in the polysaccharide biosynthesis. In H.influenzae and E. coli, however, these proteins are not associated withcapsular gene clusters. The analysis of isogenic mutants impaired in theexpression of Orf2X should give more insight in the presumed role ofOrf2X in the polysaccharide biosynthesis of S. suis type 2.

The gene products encoded by the cps2E, cps2F, cps2G, cps2H, cps2J andcps2K genes showed little similarity with glycosyltransferases ofseveral Gram-positive or Gram-negative bacteria (18, 19, 20, 22, 25).The cps2E gene product shows some similarity with the Cps14E protein ofS. pneumoniae (18, 19). CpS14E is a glucosyl-1-phosphate transferasethat links glucose to a lipid carrier (18). In S. pneumoniae this is thefirst step in the biosynthesis of the oligosaccharide repeating unit.The structure of the S. suis serotype 2 capsule contains glucose,galactose, rhamnose, N-acetyl glucosamine and sialic acid in a ratio of3:1:1:1:1 (7). Based on these data, we conclude that Cps2E of S. suishas glucosyltransferase activity and is involved in the linkage of thefirst sugar to the lipid carrier.

The C-terminal region of the cps2F gene product showed some similaritywith the RfbU of Salmonella enteritica. RfbU was shown to havemannosyltransferase activity (24). Because mannosyl is not a componentof the S. suis type 2 polysaccharide, a mannosyltransferase activity isnot expected in this organism. Nevertheless, cps2F encodes aglycosyltransferase with another sugar specificity.

Cps2G showed moderate similarity to a family of gene products suggestedto encode galactosyltransferase activities (22, 24, 40). Hence, asimilar activity is shown for Cps2G.

Cps2H showed some similarity with LgtD of H. influenzae (U32768).Because LgtD was proposed to have glycosyltransferase activity, asimilar activity is fulfilled by Cps2H.

Cps2J and Cps2K showed similarity to Cps14J of S. pneumoniae (20). Cps2Jshowed similarity with Cps14I of S. pneumoniae as well. Cps14I was shownto have N-acetyl glucosaminyltransferase activity, whereas Cps14J has aβ-1,4-galactosyltransferase activity (20). In S. pneumoniae, Cps14I isresponsible for the addition of the third sugar and Cps14J for theaddition of the last sugar in the synthesis of the type 14 repeatingunit (20). Because the capsule of S. suis type 2 contains galactose aswell as N-acetyl glucosamine components, galactosyltransferase as wellas N-acetyl glucoaminyltransferase activities could be envisaged for thecps2J and cps2K gene products, respectively. As was observed for Cps14Iand Cps14J, the N-termini of Cps2J and Cps2K showed a significant degreeof sequence similarity. Within the N-terminal domains of Cps14I andCps14J, two small regions were identified, which were also conserved inseveral other glycosyltransferases (22). Within these two regions, twoAsp residues were proposed to be important for catalytic activity. Thetwo conserved regions, DXS and DXDD, were also found in Cps2J and Cps2K.

The function of Cps2I remains unclear. Cps2I showed some similarity witha protein of A. actinomycetemcomitans. Although this protein part is ofthe gene cluster responsible for the serotype-B-specific antigens, thefunction of the protein is unknown.

We further describe the identification and characterization of the cpsgenes specific for S. suis serotypes 1, 2 and 9. After the entire cps2locus of S. suis serotype 2 was cloned and characterized, functions formost of the cps2 gene products could be assigned by sequence homologies.Based on these data, the glycosyltransferase activities, required fortype specificity, could be located in the center of the operon.Cross-hybridization experiments, using the individual cps2 genes asprobes on chromosomal DNAs of the 35 different serotypes, confirmed thisidea. The regions containing the type-specific genes of serotypes 1 and9 could be cloned and characterized, showing that an identical geneticorganization of the cps operons of other S. suis serotypes exists. Thecps1E, cps1F, cps1G, cps1H, and cps1I genes revealed a strikingsimilarity with cps14E, cps14F, cps14G, cps14H and cps14J genes of S.pneumoniae. Interestingly, S. pneumoniae serotype 14 is the serotypemost commonly associated with pneumococcal infections in young children(54), whereas S. suis serotype 1 strains are most commonly isolated frompiglets younger than 8 weeks (46). In S. pneumoniae, the cps14E, cps14G,cps14I and cps14J encode the glycosyltransferases required for thesynthesis of the type 14 tetrameric repeating unit, showing that thecps1E, cps1G and cps1I genes encoded glycosyltransferases. The precisefunctions of these genes as well as the substrate specificities of theenzymes can be established. In S. pneumoniae, the cps14E gene was shownto encode a glucosyl-1-phosphate transferase catalyzing the transfer ofglucose to a lipid carrier. Moreover, cpsE-like genes were found in S.pneumoniae serotypes 9N, 13, 14, 15B, 15C, 18F, 18A and 19F (60). CpsEmutants were constructed in the serotypes 9N, 13, 14 and 15B. All mutantstrains lacked glucosyltransferase activity (60). Moreover, in all theseS. pneumoniae serotypes, the cpsE gene seemed to be responsible for theaddition of glucose to the lipid carrier. Based on these data, wesuggest that in S. suis type 1, the cps1E gene may fulfill a similarfunction. The structure of the S. suis type 1 capsule is unknown, but itis composed of glucose, galactose, N-acetyl glucosamine, N-acetylgalactosamine and sialic acid in a ratio of 1:2.4:1:1:1.4 (5).Therefore, a role of a cpsE-like glucosyltransferase activity can easilybe envisaged. CpsE-like sequences were also found in serotypes 2, 1/2and 14.

For polysaccharide biosynthesis in S. pneumoniae type 14, transfer ofthe second sugar of the repeating unit to the first lipid-linked sugaris performed by the gene products of cps14F and cps14G (20). Similar toCps14F and Cps14G, the S. suis type 1 proteins Cps1F and Cps1G may actas one glycosyltransferase performing the same reaction. Cps14F andCps14G of S. pneumoniae showed similarity to the N-terminal half andC-terminal half of the SpsK protein of Sphingomonas (20, 67),respectively. This suggests a combined function for both proteins.Moreover, cps14F- and cps14G-like sequences were found in severalserotypes of S. pneumoniae and these genes always seemed to existtogether (60). The same was observed for S. suis type 1. The cps1F andcps1G probes hybridized with type 1 and type 14 strains.

According to the similarity found between the cps1H gene and the cps14Hgene of S. pneumoniae (20), cps1H is expected to encode a polysaccharidepolymerase.

The protein encoded by the cps1I gene showed some similarity with theCps14J protein of S. pneumoniae (19). The cps14J gene was shown toencode a β-1,4-galactosyltransferase activity, responsible for theaddition of the fourth (i.e. last) sugar in the synthesis of the S.pneumoniae serotype 14 polysaccharide. In S. suis type 2, the proteinsencoded by the cps2J and cps2K genes showed similarity to the Cps14Jprotein. However, no significant homologies were found between Cps2J,Cps2K and Cps1I. In the N-terminal regions of Cps14J and Cps14I, twosmall conserved regions, DXS and DXDD, were identified (19). Theseregions seemed to be important for catalytic activity (13). At the samepositions in the sequence, Cps2I contained the regions DXS and DXED.

In the region between Cps1G and Cps1H, three small Orfs were identified.Since the Orfs were expressed in three different reading frames, and didnot contain potential start sites, expression is not expected. However,the three potential gene products encoded by this region showed somesimilarity with three successive regions of the C-terminal part of theEpsK (protein of Streptococcus thermophiles (27% identity, 40). Theregion related to the first 82 amino acids is lacking. The EpsK proteinwas suggested to play a role in the export of the exopolysaccharide byrendering the polymerized exopolysaccharide more hydrophobic through alipid modification. These data could suggest that the sequences in theregion between Cps1G and Cps1H originated from an epsK-like sequence.Hybridization experiments showed that this epsK-like region is alsopresent in other serotype 1 strains as well as in serotype 14 strains(results not shown).

The function of most of the cloned serotype 9 genes can be established.Based on sequence similarity data, the cps9E and cps9F genes could beglycosyltransferases (61, 24, 63, 64, 65). Moreover, the cps9G and cps9Hgenes showed similarity to genes located in regions involved inpolysaccharide biosynthesis, but the function of these genes is unknown(68).

Cross-hybridization experiments using the individual cps2, cps1 and cps9genes as probes showed that the cps9G and cps9H probes specificallyhybridized with serotype 9 strains. Therefore, these are useful as toolsfor the identification of S. suis type 9 strains both for diagnosticpurposes as well as in epidemiological and transmission studies. Wepreviously developed a PCR method which can be used to detect S. suisstrains in nasal and tonsil swabs of pigs (62). The method was used toidentify pathogenic (EF-positive) strains of S. suis serotype 2. BesidesS. suis type 2 strains, serotype 9 strains are frequently isolated fromorgans of diseased pigs. However, until now, a rapid and sensitivediagnostic test was not available for type 9 strains. Therefore, thetype 9-specific probes or the type 9-specific PCR is of great diagnosticvalue. The cps1F, cps1G and cps1I probes hybridized with serotype 1 aswell as with serotype 14 strains. In coagglutination tests, type 1strains react with the anti-type 1 as well as with the anti-type 14antisera (56). This suggests the presence of common epitopes betweenthese serotypes. On the other hand, type 1 strains agglutinated onlywith anti-type 1 serum (56, 57), indicating that it is possible todetect differences between those serotypes.

The cps2F, cps2G, cps2H, cps2I and cps2J probes hybridized withserotypes 2 and 1/2 only. Serotype 34 showed a weak hybridizing signalwith the cps2G probe. As shown in agglutination tests, type 1/2 strainsreact with sera directed against type 1 as well as with sera directedagainst type 2 strains (46). Therefore, type 1/2 shared antigens withboth types 1 and 2. Based on the hybridization patterns of serotype 1/2strains with the cps1- and cps2-specific genes, serotype 1/2 seemed tobe more closely related to type 2 strains than to type 1 strains. In ourcurrent studies, we identify type-specific genes, primers or probeswhich are used for the discrimination of serotypes 1, 14 and 2 and 1/2and others of the 35 serotypes yet known. Furthermore, type-specificgenes, primers or probes can now easily be developed for yet unknownserotypes, once they become isolated.

Cloning and Characterization of a Further Part of the Cps2 Locus.

Based on the established sequence, 11 genes, designated cps2L to cps2T,orf2U and orf2V, were identified. A gene homologous to genes involved inthe polymerization of the repeating oligosaccharide unit (cps20) as wellas genes involved in the synthesis of sialic acid (cps2P to cps2T) wereidentified. Moreover, hybridization experiments showed that the genesinvolved in the sialic acid synthesis are present in S. suis serotypes1, 2, 14, 27 and 1/2. The “cps2M” and “cps2N” regions showed similarityto proteins involved in the polysaccharide biosynthesis of otherGram-positive bacteria. However, these regions seemed to be truncated orwere non-functional as the result of frame-shift or point mutations. Atits 3′-end, the cps2 locus contained two insertional elements (“orf2U”and “orf2V”), both of which seemed to be non-functional.

To clone the remaining part of the cps2 locus, sequences of the 3′-endof pCPS26 (FIG. 1, part C) were used to identify a chromosomal fragmentcontaining cps2 sequences located further downstream. This fragment wascloned in pKUN19, resulting in pCPS29. Using a similar approach, wesubsequently isolated the plasmids pCPS30 and pCPS34 containingdownstream cps2 sequences (FIG. 1, part C).

Analysis of the Cps2 Operon.

The complete nucleotide sequence of the cloned fragments was determined.Examination of the compiled sequence revealed the presence of: asequence encoding the C-terminal part of Cps2K, six apparentlyfunctional genes (designated cps2O-cps2T) and the remnants of 5different ancestral genes (designated “cps2L,” “cps2M,” “cps2N,” “orf2U”and “orf2V”). The latter genes seemed to be truncated or incomplete asthe result of the presence of stop codons or frame-shift mutations (FIG.1, part A). Neither potential promoter sequences nor potential stem-loopstructures could be identified within the sequenced region. Aribosome-binding site precedes each ORF and the majority of the ORFs arevery closely linked. Three intergenic gaps were found: one between“cps2M” and “cps2N” (176 nucleotides), one between cps2O and cps2P (525nucleotides), and one between cps2T and “orf2U” (200 nucleotides). Theseand our above data show that Orf2X and Cps2A-Orf2T are part of a singleoperon.

A list of all loci and their properties is shown in Table 4. The “cps2L”region contained three potential ORFs of 103, 79 and 152 amino acids,respectively, which were only separated from each other by stop codons.Only the first ORF is preceded by a potential ribosomal binding site andcontained a methionine start codon. This suggests that “cps2L”originates from an ancestral cps2L gene, which coded for a protein of339 amino acids. The function of this hypothetical cps2L protein remainsunclear so far: no significant homologies were found between Cps2L andproteins present in the data libraries. It is not clear whether thefirst ORF of the “cps2L” region is expressed into a protein of 103 aminoacids. The “cps2M” region showed homology to the N-terminal 134 aminoacids of the NeuA proteins of Streptococcus agalactiae and Escherichiacoli (AB017355, 32). However, although the “cps2M” region contained apotential ribosome binding site, a methionine start codon was absent.Compared with the S. agalactiae sequence, the ATG start codon wasreplaced by a lysin encoding AAG codon. Moreover, the region homologousto the first 58 amino acids of the S. agalactiae NeuA (identity 77%) wasseparated from the region homologous to amino acids 59-134 of NeuA by arepeated DNA sequence of 100-bp (see herein). In addition, the regionhomologous to amino acids 59 to 95 of NeuA (identity 32%) and the regionhomologous to the amino acids 96 to 134 of NeuA (identity 50%) werepresent in different reading frames. Therefore, the partial andtruncated NeuA homologue is probably nonfunctional in S. suis. The“cps2N” region showed homology to CpsJ of S. agalactiae (accession no.AB017355). However, sequences homologous to the first 88 amino acids ofCpsJ were lacking in S. suis. Moreover, the homologous region waspresent in two different reading frames. The protein encoded by thecps2O gene showed homology to proteins of several streptococci involvedin the transport of the oligosaccharide repeating unit (accession no.AB017355), suggesting a similar function for Cps2O. The proteins encodedby the cps2P, cps2S and cps2T genes showed homology to the NeuB, NeuDand NeuA proteins of S. agalactiae and E. coli (accession no AB017355).Because the “cps2M” region also showed homology to NeuA of E. coli, theS. suis cps2 locus contains a functional neuA gene (cps2T) as well as anonfunctional (“cps2M”) gene. The mutual homology between these tworegions showed an identity of 77% at the amino acid level over aminoacids 1-58 and 49% over the amino acids 59-134. Cps2Q and Cps2R showedhomology to the N-terminal and C-terminal parts of the NeuC protein ofS. agalactiae and E. coli, respectively. This suggests that the functionof the S. agalactiae NeuC protein in S. suis is likely fulfilled by twodifferent proteins. In E. coli, the neu genes are known to be involvedin the synthesis of sialic acid. NeuNAc is synthesized fromN-acetylmannosamine and phosphoenolpyruvate by NeuNAc synthetase.Subsequently, NeuNAc is converted to CMP-NeuNAc by the enzyme CMP-NeuNAcsynthetase. CMP-NeuNAc is the substrate for the synthesis ofpolysaccharide. In E. coli, K1 NeuB is the NeuNAc synthetase, and NeuAis the CMP-NeuNAc synthetase. NeuC has been implicated in the NeuNAcsynthesis, but its precise role is not known. The precise role of NeuDis not known. A role of the Cps2P-Cps2T proteins in the synthesis ofsialic acid can easily be envisaged, since the capsule of S. suisserotype 2 is rich in sialic acid. In S. agalactiae, sialic acid hasbeen shown to be critical to the virulence function of the type IIIcapsule. Moreover, it has been suggested that the presence of sialicacid in the capsule of bacteria which can cause meningitis may beimportant for these bacteria to breach the blood-brain barrier. So far,however, the requirement of the sialic acid for virulence of S. suisremains unclear.

“Orf2U” and “Orf2V” showed homology to proteins located on two differentinsertional elements. “Orf2U” is homologous to IS1194 of Streptococcusthermophilus, whereas “Orf2V” showed homology to a putative transposaseof Streptococcus pneumoniae. This putative transposase was recentlyfound to be associated with the type 2 capsular locus of S. pneumoniae.Compared with the original insertional elements in S. thermophilus andS. pneumoniae, both “Orf2U” and “Orf2V” are likely to be non-functionaldue to frame shift mutations within their coding regions.

A striking observation was the presence of a sequence of 100 bp (FIG.10) which was repeated three times within the cps2 operon. The sequenceis highly conserved (between 94% and 98%) and was found in theintergenic regions between cps2G and cps2H, within “cps2M” and betweencps2O and cps2P. No significant homologies were found between this100-bp direct repeat sequence and sequences present in the datalibraries, suggesting that the sequence is unique for S. suis.

Distribution of the Cps2 Sequences Among the 35 S. suis Serotypes.

To examine the presence of sialic acid encoding genes in other S. suisserotypes, we performed cross-hybridization experiments. DNA fragmentsof the individual cps2 genes were amplified by PCR, radiolabeled with32P and hybridized to chromosomal DNA of the reference strains of the 35different S. suis serotypes. As a positive control, we used a probespecific for S. suis 16S rRNA. The 16S rRNA probe hybridized with almostequal intensities to all serotypes tested (Table 4). The “cps2L”sequence hybridized with DNA of serotypes 1, 2, 14 and 1/2. The “cps2M”,cps2O, cps2P, cps2Q, cps2R, cps2S and cps2T genes hybridized with DNA ofserotypes 1, 2, 14, 27 and 1/2. Because the cps2P-cps2T genes are mostlikely involved in the synthesis of sialic acid, these results suggestthat sialic acid is also a part of the capsule in the S. suis serotypes1, 2, 14, 27 and 1/2. This is in agreement with the finding that theserotypes 1, 2 and 1/2 possess a capsule that is rich in sialic acid.Although the chemical compositions of the capsules of serotypes 14 and27 are unknown, recent agglutination studies using sialic acid-bindinglectins suggested the presence of sialic acid in S. suis serotype 14,but not in serotype 27. In these studies, sialic acid was also detectedin serotypes 15 and 16. Since the latter observation is not in agreementwith our hybridization studies, it might be that other genes, nothomologous to the cps2P-cps2T genes, are responsible for the sialic acidsynthesis in serotypes 15 and 16.

A probe based on “cps2N” sequences hybridized with DNA from serotypes 1,2, 14 and 1/2. A probe specific for “orf2U” hybridized with serotypes 1,2, 7, 14, 24, 27, 32, 34, and 1/2, whereas a probe specific for “orf2V”hybridized with many different serotypes. In addition, we prepared aprobe specific for the 100-bp direct repeat sequence. This probehybridized with the serotypes 1, 2, 13, 14, 22, 24, 27, 29, 32, 34 and1/2 (Table 4). To analyze the number of copies of the direct repeatsequence within the S. suis serotype 2 chromosome, a Southern blothybridization and analysis was performed. Therefore, chromosomal DNA ofS. suis serotype 2 was digested with NcoI and hybridized with a32P-labeled direct repeat sequence. Only one hybridizing fragment,containing the three direct repeats present on the cps2 locus, was found(results not shown). This indicates that the 100-bp direct repeatsequence is only associated with the cps2 locus. In S. pneumoniae, a115-bp long repeated sequence was found to be associated with thecapsular genes of serotypes 1, 3, 14 and 19F. In S. pneumoniae, this115-bp sequence was also found in the vicinity of other genes involvedin pneumococcal virulence (hyaluronidase and neuraminidase genes). Aregulatory role of the 115-bp sequence in coordinate control of thesevirulence-related genes was suggested.

To study the role of the capsule in resistance to phagocytosis and invirulence, we constructed two isogenic mutants in which capsulesynthesis was disturbed. In 10cpsB, the cps2B gene was disturbed by theinsertion of an antibiotic-resistance gene, whereas in 10cpsEF, parts ofthe cps2S and cps2F genes were replaced. Both mutant strains seemed tobe completely unencapsulated. Because the cps2 genes seemed to be partof an operon, polar effects cannot be excluded. Therefore, these datadid not give any information about the role of Cps2B, Cps2E or Cps2F inthe polysaccharide biosynthesis. However, the results clearly show thatthe capsular polysaccharide of S. suis type 2 is a surface componentwith antiphagocytic activity. In vitro wild-type encapsulated bacteriaare ingested by phagocytes at a very low frequency, whereas the mutantunencapsulated bacteria are efficiently ingested by porcine macrophages.Within 2 hours, over 99.6% of mutant bacteria were ingested and over 92%of the ingested bacteria were killed. Intracellularly, wild-type as wellas mutant strains seemed to be killed with the same efficiency. Thissuggests that the loss of capsular material is associated with loss ofcapacity to resist uptake by macrophages. This loss of resistance to invitro phagocytosis was associated with a substantial attenuation of thevirulence in germfree pigs. All pigs inoculated with the mutant strainssurvived the experiment and did not show any specific clinical signs ofdisease. Only some aspecific clinical signs of disease could beobserved. Moreover, mutant bacteria could be reisolated from the pigs.This supports the idea that, as in other pathogenic Streptococci, thecapsule of S. suis acts as an important virulence factor. Transposonmutants prepared by Charland impaired in the capsule production showed areduced virulence in pigs and mice. To construct these mutants, the type2 reference strain S735 was used. We previously showed that this strainis only weakly virulent for young pigs. Moreover, the insertion site ofthe transposon is unsolved so far.

As a Further Example Herein a Rapid PCT Test for Streptococcus suis Type7 is Described.

Recent epidemiological studies on Streptococcus suis infections in pigsindicated that, besides serotypes 1, 2 and 9, serotype 7 is alsofrequently associated with diseased animals. For the latter serotype,however, no rapid and sensitive diagnostic methods are available. Thishampers prevention and control programs. Here we describe thedevelopment of a type-specific PCR test for the rapid and sensitivedetection of S. suis serotype 7. The test is based on DNA sequences ofcapsular (cps) genes specific for serotype 7. These sequences could beidentified by cross-hybridization of several individual cps genes withthe chromosomal DNAs of 35 different S. suis serotypes.

Streptococcus suis is an important cause of meningitis, septicemia,arthritis and sudden death in young pigs [69, 70]. It can, however, alsocause meningitis in man (71). Attempts to control the disease are stillhampered by the lack of sufficient knowledge about the epidemiology ofthe disease and the lack of effective vaccines and sensitivediagnostics.

S. suis strains can be identified and classified by their morphological,biochemical and serological characteristics (70, 73, 74). Serologicalclassification is based on the presence of specific antigenicdeterminants. Isolated and biochemically characterized S. suis cells areagglutinated with a panel of specific sera. These typing methods arevery laborious and time-consuming and can only be performed on isolatedcolonies. Moreover, it has been reported that nonspecificcross-reactions may occur among different types of S. suis (75, 76).

So far, 35 different serotypes have been described (7, 78, 79). S. suisserotype 2 is the most prevalent type isolated from diseased pigs,followed by serotypes 9 and 1. However, recently, serotype 7 strainswere also frequently isolated from diseased pigs (80, 81, 82). Thissuggests that infections with S. suis serotype 7 strains seemed to be anincreasing problem. Moreover, the virulence of S. suis serotype 7strains was confirmed by experimental infection of young pigs (83).

Recently, rapid and sensitive PCR assays specific for serotypes 2 (and1/2), 1 (and 14) and 9 were developed (84). These assays were based onthe cps loci of S. suis serotypes 2, 1 and 9 (84, 85). However, untilnow, no rapid and sensitive diagnostic test was available for S. suisserotype 7. Herein we describe the development of a PCR test for therapid and sensitive detection of S. suis serotype 7 strains. The test isbased on DNA sequences which form a part of the cps locus of S. suisserotype 7. Compared with the serological serotyping methods, the PCRassay was a rapid, reliable and sensitive assay. Therefore, this test,in combination with the PCR tests which we previously developed forserotypes 1, 2 and 9, will undoubtedly contribute to a more rapid andreliable diagnosis of S. suis and may facilitate control and eradicationprograms.

Materials and Methods Bacterial Strains, Growth Conditions andSerotyping.

The bacterial strains and plasmids used in this study are listed inTable 7. The S. suis reference strains were obtained from M. Gottschalk,Canada. S. suis strains were grown in Todd-Hewitt broth (code CM189,Oxoid), and plated on Columbia agar blood base (code CM331, Oxoid)containing 6% (v/v) horse blood. E. coli strains were grown in Luriabroth (86) and plated on Luria broth containing 1.5% (w/v) agar. Ifrequired, ampicillin was added to the plates. The S. suis strains wereserotyped by the slide agglutination test with serotype-specificantibodies (70).

DNA Techniques.

Routine DNA manipulations and PCR reactions were performed as describedby Sambrook et al. (88). Blotting and hybridization were performed asdescribed previously (84, 86).

DNA Sequence Analysis.

DNA sequences were determined on a 373A DNA Sequencing system (AppliedBiosystems, Warrington, GB). Samples were prepared by use of anABI/PRISM dye terminator cycle sequencing ready reaction kit (AppliedBiosystems). Custom-made sequencing primers were purchased from LifeTechnologies. Sequencing data were assembled and analyzed using theMcMollyTetra program. The BLAST program was used to search for proteinsequences homologous to the deduced amino acid sequences.

PCR.

The primers used for the cps7H PCR correspond to the positions 3334-3354and 3585-3565 in the S. suis cps7 locus. The sequences were:5′-AGCTCTAACACGAAATAAGGC-3′ (SEQ ID NO:7) and5′-GTCAAACACCCTGGATAGCCG-3′ (SEQ ID NO:8).

The reaction mixtures contained 10 mM Tris-HCl, pH 8.3; 1.5 mM MgCl2; 50mM KCl; 0.2 mM of each of the four deoxynucleotide triphosphates; 1microM of each of the primers and 1 U of AmpliTaq Gold DNA polymerase(Perkin Elmer Applied Biosystems, New Jersey). DNA amplification wascarried out in a Perkin Elmer 9600 thermal cycler and the programconsisted of an incubation for 10 min at 95° C. and 30 cycles of 1 minat 95° C., 2 min at 56° C. and 2 min at 72° C.

Results and Discussion Cloning of the Serotype 7-Specific Cps Genes.

To isolate the type-specific cps genes of S. suis serotype 7, we usedthe cps9E gene of serotype 9 as a probe to identify chromosomal DNAfragments of type 7 containing homologous DNA sequences (84). A 1.6-kbPstI fragment was identified and cloned in pKUN19. This yielded pCPS7-1(FIG. 11, part C). In turn, this fragment was used as a probe toidentify an overlapping 2.7 kb ScaI-ClaI fragment. pGEM7 containing thelatter fragment was designated pCPS7-2 (FIG. 11, part C).

Analysis of the Cloned Cps7 Genes.

The complete nucleotide sequences of the inserts of pCPS7-1, pCPS7-2were determined. Examination of the cps7 sequence revealed the presenceof two complete and two incomplete open reading frames (ORFs) (FIG. 11,part C). All ORFs are preceded by a ribosome-binding site. In accordwith the data obtained for the cps1, cps2 and cps9 genes of serotypes 1,2 and 9, respectively, the type 7 ORFs are very closely linked to eachother. The only significant intergenic gap was that found between cps7Eand cps7F (443 nucleotides). No obvious promoter sequences or potentialstem-loop structures were found in this region. This suggests that, asin serotypes 1, 2 and 9, the cps genes in serotype 7 form part of anoperon.

An overview of the ORFs and their properties is shown in Table 8. Asexpected on the basis of the hybridization data (84), the Cps9E andCps7E proteins showed a high similarity (identity 99%, Table 8). Basedon sequence comparisons between Cps9E and Cps7E, the PstI fragment ofpCPS7-1 lacks the region encoding the first 371 codons of Cps7E. TheC-terminal part of the protein encoded by the cps7F gene showed somesimilarity with the Bp1G protein of Bordetella pertussis (88), as wellas with the C-terminal part of S. suis Cps2E (85). Both Bp1G and Cps2Ewere suggested to have glycosyltransferase activity and are probablyinvolved in the linkage of the first sugar to the lipid carrier (85,88). The protein encoded by the cps7G gene showed similarity with theBlpF protein of Bordetella pertussis (88). BlpF is likely to be involvedin the biosynthesis of an amino sugar, suggesting a similar function forCps7G. The protein encoded by the cps7H gene showed similarity with theWbdN protein of E. coli (89) as well as with the N-terminal part of theCps2K protein of S. suis (81). Both WbdN and Cps2K were suggested tohave glycosyltransferase activity (85, 89).

Serotype 7-Specific Cps Genes.

To determine whether the cloned fragments in pCPS7-1 and pCPS7-2contained serotype 7-specific DNA sequences, cross-hybridizationexperiments were performed. DNA fragments of the individual cps7 geneswere amplified by PCR, labeled with 32P, and used to probe spot blots ofchromosomal DNA of the reference strains of 35 different S. suisserotypes. The results are summarized in Table 9. As expected, based onthe data obtained with the cps9E probe (84), the cps7E probe hybridizedwith chromosomal DNA of many different S. suis serotypes. The cps7F andcps7G probes showed hybridization with chromosomal DNA of S. suisserotypes 4, 5, 7, 17, and 23. However, the cps7H probe hybridized withchromosomal DNA of serotype 7 only, indicating that this gene isspecific for serotype 7.

Type-Specific PCR.

We tested whether we could use PCR instead of hybridization for thetyping of the S. suis serotype 7 strains. For that purpose, we selectedan oligonucleotide primer set within the cps7H gene with which anamplified fragment of 251-bp was expected. In addition, we included inour analysis several S. suis serotype 7 strains, other than thereference strain. These strains were obtained from different countriesand were isolated from different organs (Table 7). The results show thatindeed a fragment of about 250-bp was amplified with all type 7 strainsused (FIG. 12, part B), whereas no PCR products were obtained withserotype 1, 2 and 9 strains (FIG. 12, part A). This suggests that thePCR test, as described here, is a rapid diagnostic tool for theidentification of S. suis serotype 7 strains. Until now, such adiagnostic test was not available for serotype 7 strains. Together withthe recently developed PCR assays for serotypes 1, 2, 1/2, 14 and 9,this assay may be an important diagnostic tool to detect pigs carryingserotype 2, 1/2, 1, 14, 9 and 7 strains and may facilitate control anderadication programs.

TABLE 1 Bacterial strains and plasmids strain/plasmid relevantcharacteristics source/reference Strain E. coli CC118 PhoA⁻ (28) XL2blue Stratagene E. coli XL2 blue Stratagene S. suis  10 virulentserotype 2 strain (49)   3 serotype 2 (63)  17 serotype 2 (63)  735reference strain serotype 2 (63) T15 serotype 2 (63) 6555 referencestrain serotype 1 (63) 6388 serotype 1 (63) 6290 serotype 1 (63) 5637serotype 1 (63) 5673 serotype 1/2 (63) 5679 serotype 1/2 (63) 5928serotype 1/2 (63) 5934 serotype 1/2 (63) 5209 reference strains serotype1/2 (63) 5218 reference strain serotype 9 (63) 5973 serotype 9 (63) 6437serotype 9 (63) 6207 serotype 9 (63) reference strains serotypes 1-34(9, 56, 14) S. suis  10 virulent serotype 2 strain (51) 10cpsB isogeniccpsB mutant of strain 10 this work 10cpsEF isogenic cpsEF mutant ofstrain 10 this work Plasmid pKUN19 replication functions pUC, Amp^(R)(23) pGEM7Zf (+) replication functions pUC, Amp^(R) Promega Corp. pIC19Rreplication functions pUC, Amp^(R) (29) pIC20R replication functionspUC, Amp^(R) (29) pIC-spc pIC19R containing spc^(R) gene of pDL282labcollection pDL282 replication functions of pBR 322 and pVT736-1,Amp^(R), (43) Spc^(R) pPHOS2 pIC-spc containing the truncated phoA geneof pPHO7 as this work a PstI-BamHI fragment pPHO7 contains truncatedphoA gene (15) pPHOS7 pPHOS2 containing chromosomal S. suis DNA thiswork pCPS6 pKUN19 containing 6 kb HindIII fragment of cps operon thiswork (FIG. 1) pCPS7 pKUN19 containing 3.5 kb EcoRI-HindIII fragment ofcps this work (FIG. 1) operon pCPS11 pCPS7 in which 0.4 kb PstI-BamHIfragment of cpsB gene this work (FIG. 1) is replaced by Spc^(R) gene ofpIC-spec pCPS17 pKUN19 containing 3.1 kb KpnI fragment of cps operonthis work (FIG. 1) pCPS18 pKUN19 containing 1.8 kb SnaBI fragment of cpsoperon this work (FIG. 1) pCPS20 pKUN19 containing 3.3 kb XbaI-HindIIIfragment of cps this work (FIG. 1) operon pCPS23 pGEM7Zf (+) containing1.5 kb MluI fragment of cps this work (FIG. 1) operon pCPS25 pIC20Rcontaining 2.5 kb KpnI-SalI fragment of pCPS17 this work (FIG. 1) pCPS26pKUN19 containing 3.0 kb HindIII fragment of cps operon this work(FIG. 1) pCPS27 pCPS25 containing 2.3 kb XbaI (blunt) - ClaI fragment ofthis work (FIG. 1) pCPS20 pCPS28 pCPS27 containing the 1.2 kb PstI-XhoISpc^(R) gene of this work (FIG. 1) pIC-spc pCPS29 pKUN19 containing 2.2kb SacI-PstI fragment of cps this work (FIG. 1) operon pCPS1-1 pKUN19containing 5 kb EcoRV fragment of cps operon this work (FIG. 1) of type1 pCPS1-2 pKUN19 containing 2.2 kb HindIII fragment of cps operon thiswork (FIG. 1) of type 1 pCPS9-1 pKUN19 containing 1 kb HindIII-XbaIfragment of cps this work (FIG. 1) operon of serotype 9 pCPS9-2 pKUN19containing 4.0 kb XbaI-XbaI fragment of cps this work (FIG. 1) operon ofserotype 9 Amp^(R): ampicillin resistant Spc^(R): spectinomycinresistant cps: capsular polysaccharide

TABLE 2 Properties of Orfs in the cps locus of S. suis serotype 2 andsimilarities to gene products of other bacteria nucleotide number ofposition in amino proposed function of gene ORF sequence acids GC %product¹ similar gene product (% identity) Orf2Z  1-719 240 44 UnknownB. subtilis YitS (26%) Orf2Y 2079-822  419 38 Transcription regulationB. subtilis YcxD (39%) Orf2X 2202-2934 244 39 Unknown H. influenzae YAAA(24%) Cps2A 3041-4484 481 39 Regulation S. pneumoniae Cps19fA (58%)Cps2B 4504-5191 229 40 Chain length determination S. pneumoniae type 3Orf1 (58%) Cps2C 5203-5878 225 40 Chain length determination/ S.pneumoniae Cps23fD (63%) Export Cps2D 5919-6648 243 38 Unknown S.pneumoniae CpsB (62%) Cps2E 6675-8052 459 33 Glycosyltransferase S.pneumoniae Cps14E (56%) Cps2F 8089-9256 389 32 Glycosyltransferase S.pneumoniae Cps23fT Cps2G  9262-10417 385 36 Glycosyltransferase S.thermophilus EpsF (25%) Cps2H 10808-12176 457 31 Glycosyltransferase S.mutans RGPEC,^(N) (29%) Cps2I 12213-13443 410 29 CP polymerase S.pneumoniae Cps23fI (48%) Cps2J 13583-14579 332 29 Glycosyltransferase S.pneumoniae Cps14J (31%) Cps2K 14574-15576 334 37 Glycosyltransferase S.pneumoniae Cps14J (40%) “Cps2L” 15618-16635 103 37 Unknown — “Cps2M”16811-17322 — 38 — S. agalactiae CpsF^(N) (77%) E. coli NeuA,^(N) (47%)“Cps2N” 17559-18342 — 39 — S. agalactiae CpsJ (43%) Cps2O 18401-19802476 40 Repeat unit transporter S. agalactiae CpsK (41%) Cps2P20327-21341 338 39 Sialic acid synthesis S. agalactiae NeuB (80%) E.coli NeuB (59%) Cps2Q 21355-21865 170 42 Sialic acid synthesis S.agalactiae NeuC^(N) (61%) E. coli NeuC^(N) (54%) Cps2R 21933-22483 18440 Sialic acid synthesis S. agalactiae NeuC^(C) (55%) E. coli NeuC^(C)(40%) Cps2S 22501-23125 208 42 Sialic acid synthesis E. coli NeuD (32%)Cps2T 23136-24366 395 40 CMP-NeuNAc synthetase S. agalactiae CpsF (49%)E. coli NeuA (34%) “Orf2U” 24566-25488 168 42 Transposase S.thermophilus IS1194 (51%) “Orf2V” 25691-26281 116 37 Transposase S.pneumoniae orf1 (85%) ¹Predicted by sequence similarity ^(N)Similarityrefers to the amino-terminal part of the gene product ^(C)Similarityrefers to the carboxy-terminal part of the gene product ORFS between “ ”are truncated or non-functional as the result of frame-shift or pointmutations

TABLE 3 Properties of ORFs in the cps gene of S. suis serotypes 1 and 9and similarities to gene products of other bacteria nucleotide numberpredicted predicted position in of amino mol. mass pI proposed functionof similar gene product (% reference/ ORF sequence G + C % acids (kDa)gene product¹ identity) accession nr. Cps1E²   1-1363 34% 454 52.2 8.0Glucosyltransferase Streptococcus suis Cps2E (26) (86%) Streptococcuspneumoniae (12) Cps14E (48%) Cps1F 1374-1821 33% 149 17.3 8.2 UnknownStreptococcus pneumoniae (14) Cps14F (83%) Cps1G 1823-2315 25% 164 19.57.5 Glycosyltransferase Streptococcus pneumoniae (14) Cps14G (50%) Cps1H3035-4202 24% 389 45.5 8.4 CP polymerase Streptococcus pneumoniae (14)Cps14H (30%) Cps1I 4917-   Glycosyltransferase Streptococcus pneumoniae(13) Cps14J (38%) Lactococcus lactis EpsG (29) (31%) Streptococcusthermophilus (28) EpsI (33%) Cps1J Glycosyltransferase Streptococcuspneumoniae (13) Cps14J (%) Cps1K³ 37% 278 32.5 7.8 GlycosyltransferaseStreptococcus pneumoniae (13) Cps14J (44%) Cps9D²  1-646 37% 215 24.98.1 Unknown Streptococcus suis Cps2D (26) (89%) Cps9E 680-  Glycosyltransferase Staphylococcus aureus Cap1D (18) (27%) Cps9F 36% 20022.3 8.2 Glycosyltransferase Staphylococcus aureus (17) Cap5M (52%)Cps9G 35% 269 31.5 8.0 Unknown Actinobacillus (AB002668_4)actinomycetemcomitans (43%) Haemophilus influenzae Lsg (O05081) (43%)Cps9H³ 30% 143 16.5 7.2 Unknown Yersinia enterolitica RfbB (33) (28%)¹Predicted by sequence similarity ²N-terminal part of protein is lacking³C-terminal part of protein is lacking

TABLE 4 Hybridization of serotype 2 cps genes and neighboring sequenceswith chromosomal DNA of serotypes serotypes 1 2 3 4 5 6 7 8 9 10 11 1213 14 15 16 17 DNA probes orf2Z + + + + + + + + + + + + ± + + + +orf2Y + + + + + + + + + + + + ± + + + + orf2X + + + + + + + + + + + +± + + + + cps2A + + + + + + + + + + + + + + + + +cps2B + + + + + + + + + + − − ± + − − ± cps2C + + + + + + + + + + + −± + − ± − cps2D + + + + + + + + + + + ± ± + − ± + cps2E + + − − − − − −− − − − − + − − − cps2F − + − − − − − − − − − − − − − − − cps2G − + − −− − − − − − − − − − − − − cps2H − + − − − − − − − − − − − − − − − cps2I− + − − − − − − − − − − − − − − − cps2J − + − − − − − − − − − − − − − −− cps2K + + − − − − − − − − − − − + − − − “cps2L” + + − − − − − − − − −− − + − − − “cps2M” + + − − − − − − − − − − − + − − − cps2N” + + − − − −− − − − − − − + − − − cps2O + + − − − − − − − − − − − + − − − cps2P + +− − − − − − − − − − − + − − − cps2Q + + − − − − − − − − − − − + − − −cps2R + + − − − − − − − − − − − + − − − cps2S + + − − − − − − − − − −− + − − − cps2T + + − − − − − − − − − − − + − − − “orf2U” + + − − − − +− − − − − − + − − − “orf2V” + + ± ± ± − ± − − − − − − + + − + 100-bprepeat + + − − − − − − − − − − + + − − −16SrRNA + + + + + + + + + + + + + + + + + serotypes 18 19 20 21 22 23 2425 26 27 28 29 30 31 32 33 34 ½ DNA probes orf2Z + + − + − + + +− + + + + + − − − + orf2Y + + ± + ± + + + + + + + + + − − − + orf2X + +− + − + + + − + + + + + − − − + cps2A + + − + − + + + − + + + + + − −− + cps2B ± ± − ± − + + + − − − + ± + − ± − + cps2C − − − − − + + + − +± − − + − ± − + cps2D + + − ± − + + + − + + + ± + − − − + cps2E − − − −− − − − − + − − − − − − − + cps2F − − − − − − − − − − − − − − − − − +cps2G − − − − − − − − − − − − − − − − ± + cps2H − − − − − − − − − − − −− − − − − + cps2I − − − − − − − − − − − − − − − − − + cps2J − − − − − −− − − − − − − − − − − + cps2K − − − − − − − − − − − − − − − − − +“cps2L” − − − − − − − − − − − − − − − − − + “cps2M” − − − − − − − − − +− − − − − − − + cps2N” − − − − − − − − − − − − − − − − − + cps2O − − − −− − − − − + − − − − − − − + cps2P − − − − − − − − − + − − − − − − − +cps2Q − − − − − − − − − + − − − − − − − + cps2R − − − − − − − − − + − −− − − − − + cps2S − − − − − − − − − + − − − − − − − + cps2T − − − − − −− − − + − − − − − − − + “orf2U” − − − − − − + − − + − − − − + − + +“orf2V” + ± − − ± + − − + − − − − + + − ± + 100-bp − − − − + − + − − + −− − − + − + + repeat 16SrRNA + + + + + + + + + + + + + + + + + +

TABLE 5 Hybridization of serotypes 1 and 9 cps genes with chromosomalDNA of other S. suis serotypes DNA probes Serotype cps1E cps1F cps1Gcps1H cps1I cps9E cps9F cps9G cps9H 16rRNA 1 + + + + + − − − − − 2 + − −− − − − − − + 3 − − − + − + − − − + 4 − − − + − + − − − + 5 − − − + − +− − − + 6 − − − − − − − − − + 7 − − − + − + − − − + 8 − − − − − − − −− + 9 − − − + − + + + + + 10 − − − + − + + − − + 11 − − − + − + ± − − +12 − − − ± − + ± − − + 13 − − − + − + − − − + 14 + + + + + − − − − + 15− − − − − − − − − + 16 − − − − − − − − − + 17 − − − + − + − − − + 18 − −− + − + − − − + 19 − − − + − + − − − + 20 − − − − − − − − − + 21 − − − +− + ± − − + 22 − − − − − − − − − + 23 − − − + − + − − − + 24 − − − +− + + − − + 25 − − − − − − − − − + 26 − − − − − − ± − − + 27 + − − − − −− − − + 28 − − − + − + ± − − + 29 − − − + − + − − − + 30 − − − + − + ± −− + 31 − − − + − + − − − + 32 − − − − − − − − − + 33 − − − − − − ± − − +34 − − − − − − − − − + ½ + − − − − − − − − +

TABLE 6 Virulence of wild-type and capsular mutant S. suis strains ingermfree pigs clinical index of the group isolation of S. suis in S.suis pigs/ mortality² morbidity³ spec non-spec. fever leucocyte pigs (n)per group in strains¹ group (n) (%) (%) symptoms⁵ symptoms⁶ index⁷index⁸ CNS serosae joints 10 4 100 100 11 88 43 44 2 3 4 10cpsB 4 0 0 010 1 3 1 3 2 10cpsEF 4 0 0 0 0 1 0 1 3 2 ¹strain10 in the wild-typestrain, strains 10cpsB and 10cpsEF are isogenic capsular mutant strains²piglets which died spontaneously or had to be killed for animal welfarereasons ³only considering pigs with specific symptoms ⁴clinical index: %of observations which matched the described criteria ⁵specific symptoms:ataxia, lameness on at lest one joint, stiffness ⁶non-specific symptoms:inappetance, depression ⁷% of observations in the experimental groupwith a body temperature >40° C. ⁸% of blood samples in the group inwhich number of granulocytes >10¹⁰/1

TABLE 7 Bacterial strains and plasmids strain/plasmid relevantcharacteristics Strain E. coli XL2 blue S. suis reference strainsserotypes 1-34 5667 serotype 7, tonsil (1993) 7037 serotype 7, organs(1994) 7044 serotype 7, brains (1994) 7068 serotype 7 (1994) 7646serotype 7 (1994) 7744 serotype 7, lungs (1996) 7759 serotype 7, joints(1996) 8169 serotype 7 (1997) 15913 serotype 7, meninges (1998) PlasmidpKUN19 replication functions pUC, Amp^(R) pGEM7Zf (+) replicationfunctions pUC, Amp^(R) pCPS9-1 pKUN19 containing 1 kb HindIII-XbaIfragment of cps operon of serotype 9 pCPS9-2 pKUN19 containing 4.09 kbXbaI-XbaI fragment of cps operon of serotype 9 pCPS7-1 pKUN19 containing1.6-kb PstI fragment of cps operon of type 7 pCPS7-2 pGEM7 containing2.7-kb ScaI-ClaI fragment of cps operon of type 7 Amp^(R): ampicillinresistant cps: capsular polysaccharide

TABLE 8 Properties of Orfs in the cps genes of S. suis serotype 7 andsimilarities to gene products of other bacteria nucleotide positionproposed function similar gene Orf in sequence of gene product product(% identity) Cps7E  1-719 Glycosyltransferase Streptococcus suis Cps9E(99%) Cps7F 1164-1863 Glycosyltransferase Bordetella pertussis BplG¹(43%) Streptococcus suis Cps2E¹ (33%) Cps7G 1872-3086 Biosynthesis aminosugar Bordetella pertussis BplF (48%) Cps7H 3104-3737Glycosyltransferase Escherichia coli WbdN (35%) Streptococcus suisCps2K² (31%) ¹similarity refers to the C-terminal part of the geneproduct ²similarity refers to the N-terminal part of the gene product

TABLE 9 Hybridization of serotype 7 cps probes with chromosomal DNA ofS. suis serotypes serotypes 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18DNA probes cps7E − − + + + − + − + + + + + + − − + + cps7F − − − + + − +− − − − − − − − − + − cps7G − − − + + − + − − − − − − − − − + − cps7H −− − − − − + − − − − − − − − − − −16SrRNA + + + + + + + + + + + + + + + + + + serotypes 19 20 21 22 23 2425 26 27 28 29 30 31 32 33 34 ½ DNA probes cps7E + − + − + + − − −− + + + − − − − cps7F − − − − + − − − − − − − − − − − − cps7G − − − − +− − − − − − − − − − − − cps7H − − − − − − − − − − − − − − − − −16SrRNA + + + + + + + + + + + + + + + + +

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1. An isolated or recombinant nucleic acid encoding a capsular genecluster of Streptococcus suis or a gene or gene fragment derivedtherefrom.
 2. The isolated or recombinant nucleic acid of claim 1,wherein the nucleic acid encodes a Streptococcus suis serotype-specificcentral region.
 3. The isolated or recombinant nucleic acid of claim 1,wherein the isolated or recombinant nucleic acid is hybridized to asecond nucleic acid encoding a gene derived from a Streptococcus suisserotype 1, 2, or 9 capsular gene cluster.
 4. An isolated or recombinantnucleic acid encoding a capsular gene cluster of Streptococcus suisserotype 2 or a gene or gene fragment derived therefrom, wherein theisolated or recombinant nucleic acid comprises SEQ ID NO: 9 and theisolated or recombinant nucleic acid encodes a capsular gene cluster ofStreptococcus suis serotype 2 or a gene or gene fragment derivedtherefrom is selected from the group of sequences consisting of SEQ IDNO: 10, SEQ ID NO: 53, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ IDNO:24, SEQ ID NO:25, SEQ ID NO: 26, SEQ ID NO:27, and SEQ ID NO:
 28. 5.An isolated or recombinant nucleic acid encoding a capsular gene clusterof Streptococcus suis serotype 1 or a gene or gene fragment derivedtherefrom, wherein the isolated or recombinant nucleic acid is SEQ IDNO:29 and the isolated or recombinant nucleic acid encodes a capsulargene cluster of Streptococcus suis serotype 1 or a gene or gene fragmentderived therefrom is selected from the group consisting of SEQ ID NO:30,SEQ ID NO: 31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35and SEQ ID NO:36.
 6. An isolated or recombinant nucleic acid encoding acapsular gene cluster of Streptococcus suis serotype 9 or a gene or genefragment derived therefrom, wherein the nucleic acid comprises SEQ IDNO:37 and wherein the isolated or recombinant nucleic acid encodes acapsular gene cluster of Streptococcus suis serotype 9 or a gene or genefragment derived therefrom is selected from the group consisting of SEQID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, and SEQ ID NO:42. 7.A nucleic acid probe or primer derived from the isolated or recombinantnucleic acid of claim 1, wherein the nucleic acid probe or primer allowsspecies or serotype specific detection of Streptococcus suis.
 8. Thenucleic acid probe or primer of claim 7, wherein the nucleic acid probeor primer further comprises at least one reporter molecule.
 9. Adiagnostic test kit comprising the nucleic acid probe or primer of claim7.
 10. A peptide encoded by the isolated or recombinant nucleic acid ofclaim
 1. 11. The peptide of claim 10, wherein the peptide is capable ofpolysaccharide biosynthesis.
 12. A process for producing a Streptococcussuis capsular antigen, the process comprising: utilizing the peptide ofclaim 11 to prepare the Streptococcus suis capsular antigen.
 13. AStreptococcus suis capsular antigen produced by the process of claim 12.14. A vaccine comprising: the Streptococcus suis capsular antigen ofclaim 13 in an amount sufficient to produce an immune response in asubject, and a suitable carrier or adjuvant.
 15. A recombinantStreptococcus suis mutant having a modified capsular gene cluster.
 16. Arecombinant microorganism comprising at least a part of a capsular genecluster of Streptococcus suis, wherein the gene cluster comprises adeletion, insertion, mutation, or point-mutation.
 17. The recombinantmicroorganism of claim 16, wherein the microorganism comprises a lacticacid bacterium.
 18. A vaccine comprising the recombinant Streptococcussuis mutant of claim
 15. 19. The vaccine of claim 18, wherein thevaccine comprises a Streptococcus mutant includes a Streptococcus mutantdeficient in capsular expression.
 20. The vaccine of claim 19, whereinthe Streptococcus mutant deficient in capsular expression is arecombinant Streptococcus mutant.
 21. The vaccine of claim 19, whereinthe Streptococcus mutant deficient in capsular expression is capable ofsurviving in an immune-competent host.
 22. The vaccine of claim 21,wherein the Streptococcus mutant deficient in capsular expression iscapable of surviving at least 4-5 days in the immune-competent host. 23.The vaccine of claim 19, wherein the Streptococcus mutant deficient incapsular expression expresses a Streptococcus virulence factor orantigenic determinant.
 24. The vaccine of claim 19, wherein theStreptococcus mutant deficient in capsular expression expresses anon-Streptococcus protein.
 25. The vaccine of claim 24 wherein thenon-Streptococcus protein has been derived from a pathogen.
 26. A methodfor controlling or eradicating a Streptococcal disease in a population,the method comprising: vaccinating subjects in the population with thevaccine of claim
 18. 27. A method for controlling or eradicating aStreptococcal disease, the method comprising: testing for the presenceof encapsulated Streptococcal strains in a sample collected from atleast one subject in a population partly or wholly vaccinated with avaccine of claim
 19. 28. A method for controlling or eradicating aStreptococcal disease comprising: testing for the presence ofcapsule-specific antibodies directed against Streptococcal strains in asample collected from at least one subject in a population partly orwholly vaccinated with a vaccine of claim
 19. 29. A method forcontrolling or eradicating a Streptococcal disease in a populationcomprising: selecting subjects in the population vaccinated with avaccine according to claim 19; and testing a sample collected from atleast one subject in the population for the presence of encapsulatedStreptococcal strains and/or for the presence of capsule-specificantibodies directed against Streptococcal strains.
 30. The nucleic acidof claim 2, wherein the serotype-specific central region encodes atleast one enzyme or fragment thereof involved in polysaccharidebiosynthesis.
 31. A vaccine comprising the microorganism of claim 16.32. The vaccine of claim 20, wherein the recombinant Streptococcusmutant has been produced by homologous recombination.
 33. The vaccine ofclaim 21, wherein the mutant is capable of surviving at least at least8-10 days in the host.